Bioluminescence Measurements Research Articles

Evaluation of the relationship between ATP bioluminescence assay and the presence of organisms associated with healthcare-associated infections

Background: High prevalence and high mortality rates associated with healthcare-associated Infections (HAI) indicate there is a need to prevent HAIs from spreading. Cleaning and disinfection of hospital surfaces are fundamental to preventing HAIs, as is the confirmation of the success of these processes. Adenosine triphosphate bioluminescence has been identified as a quicker way to confirm cleaning, but questions remain regarding its specificity regarding microorganisms important to HAIs. Methods: This study evaluated ATP bioluminescence' se fficacy in determining microbial contamination on 17 surfaces from the healthcare environment, and to determine if the ATP measurements of Acinetobacter baumannii, Candida albicans, Enterococcus faecalis, Escherichia coli, Mycobacterium smegmatis, and methicillin-resistant Staphylococcus aureus corresponded to quantitative microbiology. Results: A strong positive correlation was discovered for each of the six organisms associated with HAIs, as well as an additional 'all organisms' analysis that combined all the six organisms. Conclusion: This study demonstrated a correlation between ATP bioluminescence measurements and quantitative microbiology; however, it was not as strong at low bacterial concentrations. Additional keywords: Acinetobacter baumannii, Adenosine triphosphate (ATP), ATP measurement, Candida albicans, Enterococcus faecalis, Escherichia coli, healthcare-associated infections, Mycobacterium smegmatis, and methicillin-resistant Staphylococcus aureus.

How reliable are ATP bioluminescence meters in assessing decontamination of environmental surfaces in healthcare settings?

BackgroundMeters based on adenosine triphosphate (ATP) bioluminescence measurements in relative light units (RLU) are often used to rapidly assess the level of cleanliness of environmental surfaces in healthcare and other settings. Can such ATP measurements be adversely affected by factors such as soil and cleaner-disinfectant chemistry?ObjectiveThis study tested a number of leading ATP meters for their sensitivity, linearity of the measurements, correlation of the readings to the actual microbial contamination, and the potential disinfectant chemicals’ interference in their readings.MethodsFirst, solutions of pure ATP in various concentrations were used to construct a standard curve and determine linearity and sensitivity. Serial dilutions of a broth culture of Staphylococcus aureus, as a representative nosocomial pathogen, were then used to determine if a given meter’s ATP readings correlated with the actual CFUs. Next, various types of disinfectant chemistries were tested for their potential to interfere with the standard ATP readings.ResultsAll four ATP meters tested herein demonstrated acceptable linearity and repeatability in their readings. However, there were significant differences in their sensitivity to detect the levels of viable microorganisms on experimentally contaminated surfaces. Further, most disinfectant chemistries tested here quenched the ATP readings variably in different ATP meters evaluated.ConclusionsApart from their limited sensitivity in detecting low levels of microbial contamination, the ATP meters tested were also prone to interference by different disinfectant chemistries.

Molecular detection of bioluminescent dinoflagellates in surface waters of the Patagonian shelf during early austral summer 2008.

We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using “universal” PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.

TH-C-17A-11: Hyperthermia-Driven Immunotherapy Using Non-Invasive Radiowaves

Purpose: The sad truth is that cancer is blamed for the death of nearly one in four people in the US. Immunotherapy offers hope for stimulating cancer immunity leading to targeted killing of cancer cells and a preventative measure for cancer recurrence. Unfortunately, the clinical efficacy of immunotherapy has not yet been established, however novel approaches are being developed, including combining immunotherapy with traditional chemotherapy, radiotherapy or thermal therapy. Therapeutics such as radiofrequency (RF) ablation and select chemotherapeutics induce mild anticancer immune responses. This project seeks to enhance the immune responses stimulated by these agents by co-delivery of nanoparticle-based chemotherapeutics and immune modulators in the presence of RF induced hyperthermia. Methods: A 4T1 mouse model of breast cancer is used to test the ability of RF waves to enhance accumulation of nanoparticles in tumor tissue by increasing blood flow and extravation of nanoparticles from hyperpermeable vessels. Images of particle and cell trafficking in the tumor are captured using an integrated RF and confocal imaging system, and tumor growth is monitored by tumor bioluminescence and caliper measurements. Results: Here we demonstrate enhanced intratumoral blood flow induced by non-invasive RF waves and an increase in nanoparticle accumulation in the tumor. IL-12 is shown to have powerful anti-tumor effects leading to tumor regression and the release of Th1-biased cytokines. Doxorubicin nanoparticles combined with adjuvant nanoparticles exhibited superior antitumor effects to single agent therapy. Conclusion: RF therapy combined with nanotherapeutics is a promising approach to enhance the delivery of therapeutics to the tumor and to stimulate a tumor microenvironment that supports the development of cancer-specific immune responses. This research was supported by the National Institute of Health grant numbers U54 CA143837 and U54 CA151668, and the Kanzius Foundation.

Influence of environmental variables in the efficiency of phage therapy in aquaculture.

Aquaculture facilities worldwide continue to experience significant economic losses because of disease caused by pathogenic bacteria, including multidrug-resistant strains. This scenario drives the search for alternative methods to inactivate pathogenic bacteria. Phage therapy is currently considered as a viable alternative to antibiotics for inactivation of bacterial pathogens in aquaculture systems. While phage therapy appears to represent a useful and flexible tool for microbiological decontamination of aquaculture effluents, the effect of physical and chemical properties of culture waters on the efficiency of this technology has never been reported. The present study aimed to evaluate the effect of physical and chemical properties of aquaculture waters (e.g. pH, temperature, salinity and organic matter content) on the efficiency of phage therapy under controlled experimental conditions in order to provide a basis for the selection of the most suitable protocol for subsequent experiments. A bioluminescent genetically transformed Escherichia coli was selected as a model microorganism to monitor real-time phage therapy kinetics through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU). For all experiments, a bacterial concentration of ≈ 105 CFU ml−1 and a phage concentration of ≈ 106–8 plaque forming unit ml−1 were used. Phage survival was not significantly affected by the natural variability of pH (6.5–7.4), temperature (10–25°C), salinity (0–30 g NaCl l−1) and organic matter concentration of aquaculture waters in a temperate climate. Nonetheless, the efficiency of phage therapy was mostly affected by the variation of salinity and organic matter content. As the effectiveness of phage therapy increases with water salt content, this approach appears to be a suitable choice for marine aquaculture systems. The success of phage therapy may also be enhanced in non-marine systems through the addition of salt, whenever this option is feasible and does not affect the survival of aquatic species being cultured.

Anti-tumor effect of Shu-gan-Liang-Xue decoction in breast cancer is related to the inhibition of aromatase and steroid sulfatase expression

Ethnopharmacological relevanceShu-Gan-Liang-Xue Decoction (SGLXD), a traditional Chinese herbal formula used to ameliorate the hot flushes in breast cancer patients, was reported to have anti-tumor effect on breast cancer. Estrogen plays a critical role in the genesis and evolution of breast cancer. Aromatase and steroid sulfatase (STS) are key estrogen synthesis enzymes that predominantly contribute to the high local hormone concentrations. The present study was to evaluate the anti-tumor effect of SGLXD on estrogen receptor (ER) positive breast cancer cell line ZR-75-1, and to investigate its underlying mechanisms both in vitro and in vivo. Materials and methodsThe anti-tumor activity of SGLXD in vitro was investigated using the MTT assay. The in vivo anti-tumor effect of SGLXD was evaluated in non-ovariectomized and ovariectomized athymic nude mice. The effect of SGLXD on enzymatic activity of aromatase and STS was examined using the dual-luciferase reporter (DLR) based on bioluminescent measurements. Aromatase and STS protein level were assessed using Western blot assay. ResultsSGLXD showed dose-dependent inhibitory effect on the proliferation of ZR-75-1 cells with IC50 value of 3.40mg/mL. It also suppressed the stimulating effect on cell proliferation of testosterone and estrogen sulfates (E1S). Oral administration of 6g/kg of SGLXD for 25 days resulted in a reduction in tumor volume in non-ovariectomized and ovariectomized nude mice. The bioluminescent measurements confirmed that SGLXD has a dual-inhibitory effect on the activity of aromatase and STS. Western blot assay demonstrated that the treatment of SGLXD resulted in a decrease in aromatase and STS protein levels both in vitro and in vivo. ConclusionOur results suggested that SGLXD showed anti-tumor effect on breast cancer cells both in vitro and in vivo. The anti-tumor activity of SGLXD is related to inhibition of aromatase and STS via decreasing their expression. SGLXD may be considered as a novel treatment for ER positive breast cancer.

Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

BackgroundExpression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.Methodology/Principal FindingsThe lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Conclusions/SignificanceOur results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC50 values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows.

Low doses of flagellin-L2 multimer vaccines protect against challenge with diverse papillomavirus genotypes

Genetically modified bacterial flagellin (Fla), a Toll-like receptor-5 (TLR5) ligand, was evaluated as a fusion partner for human papillomavirus (HPV) L2-based immunogens in two animal challenge models; either cutaneous inoculation of rabbits with HPV ‘quasivirions’ containing cottontail rabbit papillomavirus (CRPV) genomes that induce warts, or intra-vaginal inoculation of mice with HPV ‘pseudovirions’ encapsidating a luciferase reporter plasmid and measurement of bioluminescence to determine infectivity. An Escherichia coli production system was developed for flagellin-L2 (Fla-L2) fusions containing either monomeric HPV-16 L2 a.a. 11(×11–200) or oligomeric L2 comprising a fusion of the a.a. 11–88 peptides of five (Fla∼5×11–88) or eight (Fla∼8×11–88) genital HPV types. Immunogenicity and bioactivity of Fla-L2 constructs were assessed using an in vitro neutralization and cell-based TLR-5 binding assay, respectively. Efficacy was evaluated following active immunization of rabbits or mice administered 3 intramuscular doses of Fla-L2 recombinants without exogenous adjuvant, followed by challenge. In addition, passive immunization studies of naïve rabbits with serial dilutions of pooled immune sera were used to determine End-Point Protection Titers (EPPT) for each formulation against a broader spectrum of HPV quasivirions. Efficacy was assessed for up to 10 weeks on the basis of wart volume induced following challenge and results compared to licensed L1-VLP vaccines (Gardasil and Cervarix). Following active immunization at doses as low as 1μg, Fla-L2 fusions afforded complete protection against infection (mice) and disease (rabbits) following either homologous or heterologous HPV challenge. Passive immunization with anti-L2 immune sera discriminated between the different vaccine candidates under evaluation, demonstrated the protective role of antibody and suggested the superiority of this oligomeric L2-TLR5 agonist fusion approach compared to L1-based vaccines in its ability to cross-protect against non-vaccine HPV types.

Bioenergetic profile analysis during maturation of embryonic stem cells

Upon withdrawal of leukemia inhibitory factor (LIF) self-renewing pluripotent mouse embryonic cells (mESC) undergo reversible commitment, followed by irreversible commitment and heterogeneous cell differentiation or apoptosis, within three days. In this study we investigated bioenergetics profile of these different maturation stages. For this purpose mESC were cultivated in the presence or absence of LIF for 24, 48 and 72h and then analysed. Mitotracker green staining showed greater mitochondrial mass in the pluripotent than in the differentiated cells. To determine if this different mitochondrial content provides bioenergetics differences between this cells populations, we measured oxygen consumption rate (OCR) as indicator of principal mitochondrial respiration parameters and extracellular acidification rate as an indicator of glycolysis using XF24 analyzer. We found that basal OCR and ECAR declined in the order: pluripotent cells (+LIF) > reversible committed cells (-LIF 24h)> irreversible committed cells (-LIF 48h) > differentiated cells (-LIF72h). This was associated with mitochondrial ATP output, estimated as coupled rate (OCR linked to ATP synthesis), showing the same downtrend. Likewise, + LIF cells possessed the highest maximal respiratory capacity indicating their higher resistance to oxidative stress comparing to maturating stages. In addition, inhibition of mitochondrial respiration impaired proliferation of +LIF, but not –LIF 72h cells. Also, suppression of mitochondrial ATP synthesis, provoked compensatory increase of glycolysis in the + LIF cells which is impaired in the course of the maturation. Bioluminescent measurement of ATP content showed the highest level in the –LIF 72h cells, implying lessen ATP turnover in the more differentiated cells. Our results demonstrated that self-renewing pluripotent mESC have bioenergetics advantage reflecting in the high OXPHOS and glycolytic activity, which declines throughout their maturation.

The central molecular clock is robust in the face of behavioural arrhythmia in aDrosophilamodel of Alzheimer's disease

Circadian behavioural deficits, including sleep irregularity and restlessness in the evening, are a distressing early feature of Alzheimer’s disease (AD). We have investigated these phenomena by studying the circadian behaviour of transgenic Drosophila expressing the amyloid beta peptide (Aβ). We find that Aβ expression results in an age-related loss of circadian behavioural rhythms despite ongoing normal molecular oscillations in the central clock neurons. Even in the absence of any behavioural correlate, the synchronised activity of the central clock remains protective, prolonging lifespan, in Aβ flies just as it does in control flies. Confocal microscopy and bioluminescence measurements point to processes downstream of the molecular clock as the main site of Aβ toxicity. In addition, there seems to be significant non-cell-autonomous Aβ toxicity resulting in morphological and probably functional signalling deficits in central clock neurons.

Quantitative bioluminescence imaging of transgene expression in intact porcine antral follicles in vitro

BackgroundThe porcine oocyte maturation in vivo occurs within the ovarian follicle and is regulated by the interactions between oocytes and surrounding follicular components, including theca, granulosa, and cumulus cells, and follicular fluid. Therefore, the antral follicle is an essential microenvironment for efficient oocyte maturation and its developmental competence. Quantitative bioluminescence imaging of firefly luciferase reporter genes in an intact antral follicle would allow investigation of changes in cellular and molecular events and in the context of the whole follicles. In this study, we investigate factors influencing bioluminescence measurements as a first step towards developing a new bioluminescence imaging system for intact antral follicles.MethodsWe analyzed the time course of bioluminescence emitted from transfected living intact follicles using a cationic lipid mediated gene transfer method with increasing doses (1-3 μg) of firefly luciferase reporter gene (pGL4). In addition, a standard luciferase assay was used to confirm the luciferase expression in granulosa cells in the transfected intact antral follicles. Finally, the dose effects of substrate, D-luciferin, were determined for optimal quantitative bioluminescence imaging of intact porcine antral follicles in vitro.ResultsThe level of luciferase activity of follicles with 3 μg pGL4 was significantly (P < 0.05) greater than the 1 μg and 2 μg groups at 1 min after D-luciferin injection. The bioluminescence intensity of transfected follicles reached a peak at 1 min, and then it was significantly (P < 0.05) reduced within 2 min after injection of D-luciferin; with the level of bioluminescence emission remained constant from 2.5 to 10 min. The bioluminescence emission was maximal with 300 μg of D-luciferin.ConclusionsThe results of this study suggested that the investigation of factors influencing bioluminescence measurements is a critical step toward developing a new bioluminescence imaging model. This study is the first to demonstrate that reporter genes can be transferred to intact granulosa cells with a lipid-mediated gene transfer method within intact follicles in vitro, and the level of transgene expression can be assessed by bioluminescence imaging in living intact antral follicles.

Efectos divergentes de la inhibición de TGF-&amp;#946; en metástasis óseas de cáncer de mama y pulmón

Work scholarship from the SEIOMM to attend the 33 Congress of the ASBMR (Toronto, Canada. 2010). Summary Background: The objective of this study lies in the determination of the validity of transforming growth factor β (TGF-β) as a therapeutic target in models of metastasis deriving from different histological types of lung cancer. Material and methods: 4-week-old immunodeficient mice inoculated with lung and breast cancer lines were treated with cytokine inhibitor peptide, control peptide or placebo. Weekly bioluminescence and microradiographic measurements were taken to determine the effects of the treatment on tumor burden and metastatic lesions in the long bones. Results: Treatment with the specific peptide against TGF-β has a protector effect in the bone of animals inoculated with the breast cancer lines, unlike what happens in the control peptide and placebo groups. However, the anti-TGF-β treatment lacks the significant therapeutic effects on the bone metastases which develop in lung cancer bearing animals. Conclusions: The role of TGF-β as a potential therapeutic target in bone metastasis is highly dependent on the histopathological type and subtype of tumor.

Bathyphotometer bioluminescence potential measurements: A framework for characterizing flow agitators and predicting flow-stimulated bioluminescence intensity

Bathyphotometer measurements of bioluminescence are used as a proxy for the abundance of luminescent organisms for studying population dynamics; the interaction of luminescent organisms with physical, chemical, and biological oceanographic processes; and spatial complexity especially in coastal areas. However, the usefulness of bioluminescence measurements has been limited by the inability to compare results from different bathyphotometer designs, or even the same bathyphotometer operating at different volume flow rates. The primary objective of this study was to compare measurements of stimulated bioluminescence of four species of cultured dinoflagellates, the most common source of bioluminescence in coastal waters, using two different bathyphotometer flow agitators as a function of bathyphotometer volume flow rate and dinoflagellate concentration. For both the NOSC and BIOLITE flow agitators and each species of dinoflagellate tested, there was a critical volume flow rate, above which average bioluminescence intensity, designated as bathyphotometer bioluminescence potential (BBP), remained relatively constant and scaled directly with dinoflagellate cell concentration. At supra-critical volume flow rates, the ratio of BIOLITE to NOSC BBP was nearly constant for the same species studied, but varied between species. The spatial pattern and residence time of flash trajectories within the NOSC flow agitator indicated the presence of dominant secondary recirculating flows, where most of the bioluminescence was detected. A secondary objective (appearing in the Appendix) was to study the feasibility of using NOSC BBP to scale flow-stimulated bioluminescence intensity across similar flow fields, where the contributing composition of luminescent species remained the same. Fully developed turbulent pipe flow was chosen because it is hydrodynamically well characterized. Average bioluminescence intensity in a 2.54-cm i.d. pipe was highly correlated with wall shear stress and BBP. This correlation, when further scaled by pipe diameter, effectively predicted bioluminescence intensity in fully developed turbulent flow in a 0.83-cm i.d. pipe. Determining similar correlations between other bathyphotometer flow agitators and flow fields will allow bioluminescence potential measurements to become a more powerful tool for the oceanographic community.

Bioluminescence Imaging Reveals Dynamics of Beta Cell Loss in the Non-Obese Diabetic (NOD) Mouse Model

We generated a mouse model (MIP-Luc-VU-NOD) that enables non-invasive bioluminescence imaging (BLI) of beta cell loss during the progression of autoimmune diabetes and determined the relationship between BLI and disease progression. MIP-Luc-VU-NOD mice displayed insulitis and a decline in bioluminescence with age which correlated with beta cell mass, plasma insulin, and pancreatic insulin content. Bioluminescence declined gradually in female MIP-Luc-VU-NOD mice, reaching less than 50% of the initial BLI at 10 weeks of age, whereas hyperglycemia did not ensue until mice were at least 16 weeks old. Mice that did not become diabetic maintained insulin secretion and had less of a decline in bioluminescence than mice that became diabetic. Bioluminescence measurements predicted a decline in beta cell mass prior to the onset of hyperglycemia and tracked beta cell loss. This model should be useful for investigating the fundamental processes underlying autoimmune diabetes and developing new therapies targeting beta cell protection and regeneration.

Characterization of a Novel Mouse Model of Multiple Myeloma and Its Use in Preclinical Therapeutic Assessment

To aid preclinical development of novel therapeutics for myeloma, an in vivo model which recapitulates the human condition is required. An important feature of such a model is the interaction of myeloma cells with the bone marrow microenvironment, as this interaction modulates tumour activity and protects against drug-induced apoptosis. Therefore NOD/SCIDγcnull mice were injected intra-tibially with luciferase-tagged myeloma cells. Disease progression was monitored by weekly bioluminescent imaging (BLI) and measurement of paraprotein levels. Results were compared with magnetic resonance imaging (MRI) and histology. Assessment of model suitability for preclinical drug testing was investigated using bortezomib, melphalan and two novel agents. Cells engrafted at week 3, with a significant increase in BLI radiance occurring between weeks 5 and 7. This was accompanied by an increase in paraprotein secretion, MRI-derived tumour volume and CD138 positive cells within the bone marrow. Treatment with known anti-myeloma agents or novel agents significantly attenuated the increase in all disease markers. In addition, intra-tibial implantation of primary patient plasma cells resulted in development of myeloma within bone marrow. In conclusion, using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma by ensuring the key interaction of tumour cells with the microenvironment.

Microwave-Assisted Extraction Combined with HPLC-MS/MS for Diagnosis of Fungal Contamination in Building Materials

Ergosterol was used as indicator of fungal contamination to assess mould presence on the building materials. Microwave-assisted saponification followed by pentane extraction was used in the sample preparation step. Samples of brick, plaster, concrete and granite were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Ergosterol concentrations in laboratory infected samples ranged from 6 to 16 ng g-1, while in samples of naturally occurring materials ranged from 66 to 3020 ng g-1. Limits of detection and of quantitation were 1.7 and 5.5 ng g-1, respectively. Recovery and matrix effect were tested using samples spiked at 100 ng g-1. Mean recovery and mean matrix effect were 65.9 and 111.6%, respectively. Microscopic observations and bioluminescence measurements were carried out for the infected samples to make additional confirmation of fungi presence. The ergosterol concentration determined by HPLC-MS/MS in building materials proved to be a suitable indicator to estimate fungal contamination. The developed method was considerably faster for this aim than bioluminescence measurements and traditionally used culture methods.

The System of Automatic Seedling Selection by the Early Molecular Diagnosis of Circadian Rhythms

Abstract We have developed the system of automatic seedling selection using an early molecular diagnosis of circadian rhythms. To measure the rhythms of gene expression by bioluminescence with in non-destructive and in real time, we used a measurement system for bioluminescence, called Kondotron. Moreover, to improve the efficiency of seedling selection, a manipulator for automatic seedling selection using the decision for the circadian rhythms in bioluminescence was introduced. Using this manipulator, it became possible to select automatically seedlings in the early stage based on the result of bioluminescent measurement. This system can measure 40 samples simultaneously more than one week in real time. Moreover, it is possible to measure seedlings continuously because new seedlings are supplied to measurement sample holders when they are picked out from the measurement holder. Therefore, it is possible to research in the flexible and wide experiment of gene expression since it can analysis the circadian rhythms under various purposes (automatic selection based on statistical processing, selection by threshold, and so on) and environment (light conditions) by this system.

Cutting Edge: FcγRIII (CD16) and FcγRI (CD64) Are Responsible for Anti-Glycoprotein 75 Monoclonal Antibody TA99 Therapy for Experimental Metastatic B16 Melanoma

mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).

Combined Inhibition of JAK2 and mTOR Signaling Results in Enhanced Efficacy in in-Vitro and Preclinical Mouse Models of JAK2V617F-Driven Myeloproliferative Disease

AbstractAbstract ▪708▪This icon denotes a clinically relevant abstract Background and Aims.The JAK1/JAK2 inhibitor Ruxolitinib (ruxo) produced rapid and sustained responses in splenomegaly and symptomatic improvement in patients (pts) with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling in myeloproliferative neoplasms (MPN). Splenomegaly and constitutional symptoms improved also after treatment with Everolimus (RAD001), a rapamycin-derivative inhibitor of the serine/threonine kinase mTOR, in a phase I/II study, pointing to mTOR pathway as a novel target for MPN therapy. BEZ235 is a dual phosphatidylinositol-3-kinase (PI3K) and mTOR inhibitor currently in clinical trials in solid cancer. Aim of the study was to investigate the efficacy of BEZ235, alone and in combination with ruxo, against MPN cells in vitro and in preclinical mouse models. Methods.We used mouse (Ba/F3 and Ba/F3-EPOR expressing wild-type (WT) or V617F(VF) mutated JAK2) and human (VF HEL and SET2 or WT K562) cell lines and primary MPN CD34+ cells from pts with MF or polycythemia vera (PV); cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status were evaluated. Effects of drug combination were analyzed according to Chou and Talalay to calculate the combination index (CI); a CI <1.0 indicates synergistic activity. For in vivo studies, two mouse models were used. (1) SCID mice receiving iv Ba/F3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on d6 to treatment groups (either drugs alone and in combination or vehicle (VE)) based on baseline luminescence. Bioluminescence measurement was done at week intervals until death. (2) A C57Bl6/J JAK2 V617F Knock-in mouse model was generated by the flex switch strategy with insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies from VF heterozygous expression. Mice were treated for 5d, then blood, spleen and bone marrow cells were analyzed. Results.We found that BEZ235 inhibited proliferation of Ba/F3 VF and Ba/F3-EPOR VF cells at concentrations significantly lower than the wt counterparts (IC50 was 64±10nM vs 10,000±500nM and 87±50nM vs 676±200nM; P<0.01); similar preferential inhibition was observed in HEL and SET2 cells compared to K562 cells (IC50, 387±90nM and 334±40nM vs 5,000±1,000nM; P<0.01). BEZ235 dose-dependently increased the percentage of cells in G0/G1 and induced apoptosis. Western blot analysis showed marked reduction of the mTOR target p4EBP1 as well as appreciable downregulation of pSTAT5 and pSTAT3 at 6 to 24h of treatment. BEZ235 impaired the proliferation of CD34+ cells from MF pts with an IC50= 43±20nM vs 780±150nM in healthy donors (P<0.01), and reduced colony formation of MF and PV hematopoietic progenitors at doses statistically lower (2 to 15-fold) than normal cells. The growth of EPO-independent colonies (EEC) in PV pts was potently inhibited (IC50=20±10nM). Co-treatment of BEZ235+ruxo resulted in synergistic inhibition of proliferation in SET2 (median CI=0.37) and BaF3-EPOR VF (CI=0.77) cells and increased apoptosis rate in SET2 (CI=0.25); drug combination was highly effective also in the EEC assay (CI=0.17). In the Ba/F3VF luc model, the median bioluminescence index (No. of pixel) on day 7 of treatment was 4.7×106 in VE animals, 7.2×106 in ruxo (P=ns), 1.1×106 in BEZ235 (P=0.04) and 4.6×105 in animals receiving the BEZ+ruxo combination (P<0.01). The median survival in mice treated with the combination of BEZ+ruxo was 30d, significantly longer than either ruxo (18.5d; P<0.01) or BEZ (24d; P<0.04) alone (15.0d in VE animals). In JAK2V617F KI mice treated for 5d, we found that drug combination was significantly more effective in reducing enlarged spleen (median spleen index (spleen weight/body weightx100): 38, 35, 27 and 7 for VE, BEZ, Ruxo and BEZ+Ruxo) and reticulocyte count (median No. per HPF: 48, 50, 44, and 3 for VE, BEZ, Ruxo and BEZ+Ruxo) than either drugs alone. The phosho levels of STAT5 and 4EBP1 in the spleen were significantly reduced in mice receiving BEZ+Ruxo as compared to single treatment. Conclusions.Combined inhibition of PI3K/mTOR and JAK2 signaling resulted in enhanced activity in preclinical models of MPN compared with either treatment alone, providing a rationale for the development of combination clinical trials. Disclosures:Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees.

Self-calibrated algorithms for diffuse optical tomography and bioluminescence tomography using relative transmission images

Reconstruction algorithms for diffuse optical tomography (DOT) and bioluminescence tomography (BLT) have been developed based on diffusion theory. The algorithms numerically solve the diffusion equation using the finite element method. The direct measurements of the uncalibrated light fluence rates by a camera are used for the reconstructions. The DOT is self-calibrated by using all possible pairs of transmission images obtained with external sources along with the relative values of the simulated data and the calculated Jacobian. The reconstruction is done in the relative domain with the cancelation of any geometrical or optical factors. The transmission measurements for the DOT are used for calibrating the bioluminescence measurements at each wavelength and then a normalized system of equations is built up which is self-calibrated for the BLT. The algorithms have been applied to a three dimensional model of the mouse (MOBY) segmented into tissue regions which are assumed to have uniform optical properties. The DOT uses the direct method for calculating the Jacobian. The BLT uses a reduced space of eigenvectors of the Green's function with iterative shrinking of the permissible source region. The reconstruction results of the DOT and BLT algorithms show good agreement with the actual values when using either absolute or relative data. Even a small calibration error causes significant degradation of the reconstructions based on absolute data.