Calcein Release Assay Research Articles

Cellular Effects and Delivery Propensity of Penetratin Is Influenced by Conjugation to Parathyroid Hormone Fragment 1-34 in Synergy with pH.

The cell-penetrating peptide (CPP) penetratin has demonstrated potential as a carrier for transepithelial delivery of cargo peptides, such as the therapeutically relevant part of parathyroid hormone, i.e., PTH(1-34). The purpose of the present study was to elucidate the relevance of pH for PTH(1-34)-penetratin conjugates and coadministered penetratin with PTH(1-34) regarding transepithelial permeation of PTH(1-34) and cellular effects. Transepithelial permeation was assessed using monolayers of the Caco-2 cell culture model, and effects on Caco-2 cellular viability kinetics were evaluated by using the Real-Time-GLO assay as well as by microscopy following Tryphan blue staining. Morphological Caco-2 cell changes were studied exploiting the impedance-based xCELLigence system as well as optically using the oCelloscope setup. Finally, the effect of pH on the folding propensity of the PTH(1-34)-penetratin conjugate and its ability to disrupt lipid membranes were assessed by circular dichroism (CD) spectroscopy and the calcein release assay, respectively. The transepithelial PTH(1-34) permeation was not pH-dependent when applying the coadministration approach. However, by applying the conjugation approach, the PTH(1-34) permeation was significantly enhanced by lowering the pH from 7.4 to 5 but also associated with a compromised barrier and a lowering of the cellular viability. The negative effects on the cellular viability following cellular incubation with the PTH(1-34)-penetratin conjugate were moreover confirmed during real-time monitoring of the Caco-2 cell viability as well as by enhanced Tryphan blue uptake. In addition, morphological changes were primarily observed for cells incubated with the PTH(1-34)-penetratin conjugate at pH 5, which was moreover demonstrated to have an enhanced membrane permeating effect following lowering of the pH from 7.4 to 5. The latter observation was, however, not a result of better secondary folding propensity at pH 5 when compared to pH 7.4.

PH regulates pore formation of a protease activated Vip3Aa from Bacillus thuringiensis

Vip3Aa insecticidal protein is produced from Bacillus thuringiensis and exerts a broad spectrum of toxicity against lepidopteran insect species. Although Vip3Aa has been effectively used as part of integrated pest management strategies, the mechanism of the toxin remains unclear. Here, we investigated the effect of pH in a range from 5.0 to 10.0 on the pore-forming activity of the trypsin activated Vip3Aa (actVip3Aa) by in vitro pore-forming assays. Based on calcein release assay, actVip3Aa could permeabilize the artificial neutral liposomes under all the pH tested, except pH10.0. The maximum membrane permeability of actVip3Aa was detected at pH8.0 and the permeability decreased and abolished when exposing to acidic and alkaline conditions, respectively. The planar lipid bilayer experiment revealed that actVip3Aa formed ion channels at pH5.0–8.0 but no current signals were detected at pH10.0, consistent with the observation from calcein release assay. The toxin formed ion channels with a diameter of 1.4nm at pH8.0 and pore size was gradually decreased when reducing the pH. This study provided a view of the molecular mechanism of Vip3Aa by which the pore-forming activity is regulated by pH.

Abstract 3641: EGFR/CD16A TandAbs are efficacious NK-cell engagers with favorable biological properties which potently kill EGFR+ tumors with and without Ras mutation

Abstract Constitutive EGFR activation plays an important role in the pathophysiology of various solid cancers, such as colorectal cancer, non-small cell lung cancer or squamous cell carcinomas of the head and neck. Tyrosine kinase inhibitors (TKI) and monoclonal antibodies (mAbs), which interfere with signal transduction and activation of EGFR, are approved for treatment of such cancers. However, intrinsic or acquired resistance to these treatments has been described for many patients. Natural killer cells (NK-cells) are important effectors of innate immunity and NK-cell engagers have shown evidence of improved safety in patients compared to T-cell engagers. To specifically utilize the cytotoxic potential of NK-cells to eliminate EGFR-expressing tumors, we developed tetravalent bispecific EGFR/CD16A TandAbs comprising fully human Fv domains recognizing human and cynomolgus EGFR and CD16A. TandAbs recognizing epitopes in the extracellular domain of EGFR differing from epitopes targeted by other mAbs were characterized. Lead candidate AFM24 shows superior cytotoxicity in terms of ADCC (main mode of action) and reduced inhibition of EGFR-mediated phosphorylation compared to cetuximab. Importantly, inhibition of EGFR-signaling is believed to contribute to skin toxicity caused by therapeutic mAbs and TKI’s. AFM24’s cytotoxic activity was tested against EGFR+ tumor cell lines including some carrying a Ras mutation, which is a negative prognostic biomarker and renders cells less susceptible to cetuximab or panitumumab. The cetuximab-resistant CRC cell line HCT-116 or the NSCLC cell line A549 (both with Ras mutations) were efficiently killed with EGFR/CD16A TandAbs in vitro. In vivo data in the HCT-116 model indicate anti-tumor efficacy of AFM24, while no efficacy of cetuximab was seen. Importantly, AFM24 does not activate NK-cells without target cell binding and does not bind to any other members of the EGFR family. While binding and cytotoxic efficacy of many therapeutic mAbs are impaired by serum IgG, no substantial change in AFM24’s binding affinity to NK-cells was observed in the presence of high concentrations of human IgG. In calcein-release cytotoxicity assays with NK-cells as effectors, we showed that the presence of IgG had only little inhibitory effect on AFM24 efficacy compared to cetuximab. In addition, competition of an anti-CD16 mAb with AFM24 in cytotoxicity assays was substantially lower than with cetuximab. Taken together our data demonstrate that AFM24 is a highly potent human antibody displaying favorable biological properties over existing mAbs. This human/cynomolgus cross-reactive agent is currently in preclinical development to treat EGFR+ malignancies and has the potential to exhibit a favorable side effect profile and reduced toxicity and to overcome resistance to other targeted anti-EGFR therapeutic agents. Citation Format: Michael Kluge, Kristina Ellwanger, Uwe Reusch, Ivica Fucek, Michael Weichel, Torsten Haneke, Stefan Knackmus, Joachim Koch, Martin Treder. EGFR/CD16A TandAbs are efficacious NK-cell engagers with favorable biological properties which potently kill EGFR+ tumors with and without Ras mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2017-3641

Development of a fully human T cell engaging bispecific antibody for the treatment of multiple myeloma.

8017 Background: Although BCMA is a plasma cell specific surface molecule attractive as an antibody target in multiple myeloma, its scarcity on the cell surface may limit the efficacy of a conventional antibody. T-cell engaging bispecific antibody approaches are highly efficacious and are particularly well suited for a membrane target with limited expression, such as BCMA. Teneobio has developed a multivalent antibody platform based on modular human VH domains, which allowed us to build T cell engaging bispecific antibodies with low and high T cell agonistic activities. Methods: UniRats were immunized with either CD3 or BCMA antigens and antigen-specific UniAbs were identified by antibody repertoire sequencing and high-throughput gene assembly, expression, and screening. High affinity binding VH sequences were selected using recombinant proteins and cells. In vitro efficacy studies included T-cell activation by cytokine- and tumor cell kill by calcein-release assays. In vivo efficacy of the molecules was evaluated in NSG mice harboring myeloma cells and human PBMCs. Results: BCMA-specific UniAbs bound plasma cells with high affinities (100-700pM) and cross-reacted with cynomolgus plasma cells. Strong and weak T cell agonists were identified that bound human T cells with high and low affinities respectively and cross-reacted with cynomolgus T cells. T cell engaging bispecifics with a strong (H929 cytotoxicity:EC50=27pM) and a weak T cell activating arm (H929 cytotoxicity: EC50=1170pM) demonstrated T-cell activation and tumor-cell cytotoxicity in vitro; bispecifics with a weak CD3 engaging arm showed markedly reduced cytokine production even at doses saturating for cytotoxicity. In viv o, BCMAxCD3 bispecific antibodies reduced tumor load and increased survival when co-administered with human PBMCs as compared to controls. Conclusions: Our results suggest that T cell engaging bispecifics with low-affinity anti-CD3 arms could be preferred for the treatment of Multiple Myeloma.

Effective antimicrobial activity of a peptide mutant Cbf-14-2 against penicillin-resistant bacteria based on its unnatural amino acids

Broad spectrum activities, a unique mode of actions and rare resistant variants make antimicrobial peptide (AMP) a potential alternative to antibiotics. However, AMPs still have limitations in clinical development due to their physiological stability, toxicity and manufacturing costs. Cbf-14, derived from cathelicidin-BF has been proven to be effective against drug-resistant bacteria. Herein, a series of Cbf-14 mutants were designed to overcome these limitations. Design strategies included substitutions of lysine (Lys) or leucine (Leu) with similar residues such as ornithine (Orn) and norleucine (Ile), which are unnatural amino acids, to generate AMPs with enhanced therapeutic potential. Antimicrobial activity, hemolytic activity and cytotoxicity against mouse spleen cells of the peptide mutants were investigated. Among all of the mutants, Cbf-14-2 was regarded as the most potent candidate with the amino acid sequence of RLLR-Orn-FFR-Orn-LKKSV-NH2, which exhibited a superior antimicrobial activity with a minimum inhibitory concentration (MIC) of 4–32μg/ml. Meanwhile, Cbf-14-2 displayed low levels of hemolysis in sheep red blood cells (sRBCs) and negligible cytotoxicity against mouse spleen cells, suggesting low toxicity against mammalian cells. A circular dichroism (CD) study indicated that Cbf-14-2 has a higher alpha-helix content than Cbf-14 (68.3% vs 35.1%) in SDS, which may contribute to its superior activity. Time-killing curves showed Cbf-14-2 can eliminate all tested bacteria within 240min, suggesting its rapid bactericidal effect. Transmission electron microscopy (TEM), flow cytometry and calcein release assays revealed its excellent antimicrobial potency by inducing membrane permeation and disruption. In addition, Cbf-14-2 (10mg/kg) could significantly elevate the survival rate of clinical strain infected mice, with a survival rate of 70.0%. Taken together, the data suggest that Cbf-14-2 possesses effective antimicrobial activity against penicillin-resistant bacteria in vitro and in vitro, thus rendering it as a potential anti-infective agent in clinical settings.

NCR1 Expression Identifies Canine Natural Killer Cell Subsets with Phenotypic Similarity to Human Natural Killer Cells.

Canines spontaneously develop many cancers similar to humans – including osteosarcoma, leukemia, and lymphoma – offering the opportunity to study immune therapies in a genetically heterogeneous and immunocompetent environment. However, a lack of antibodies recognizing canine NK cell markers has resulted in suboptimal characterization and unknown purity of NK cell products, hindering the development of canine models of NK cell adoptive immunotherapy. To this end, we generated a novel antibody to canine NCR1 (NKp46), the putative species-wide marker of NK cells, enabling purification of NK cells for further characterization. We demonstrate that CD3−/NKp46+ cells in healthy and osteosarcoma-bearing canines have phenotypic similarity to human CD3−/NKp46+ NK cells, expressing mRNA for CD16 and the natural cytotoxicity receptors NKp30, NKp44, and NKp80. Functionally, we demonstrate with the calcein release assay that canine CD3−/NKp46+ cells kill canine tumor cell lines without prior sensitization and secrete IFN-γ, TNF-α, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor as measured by Luminex. Similar to human NK cells, CD3−/NKp46+ cells expand rapidly on feeder cells expressing 4-1BBL and membrane-bound IL-21 (median = 20,283-fold in 21 days). Furthermore, we identify a minor Null population (CD3−/CD21−/CD14−/NKp46−) with reduced cytotoxicity against osteosarcoma cells, but similar cytokine secretion as CD3−/NKp46+ cells. Null cells in canines and humans have reduced expression of NKG2D, NKp44, and CD16 compared to NKp46+ NK cells and can be induced to express NKp46 with further expansion on feeder cells. In conclusion, we have identified and characterized canine NK cells, including an NKp46− subset of canine and human NK cells, using a novel anti-canine NKp46 antibody, and report robust ex vivo expansion of canine NK cells sufficient for adoptive immunotherapy.

Effect of fatty acids on the permeability barrier of model and biological membranes

Because of the amphipathicity and conical molecular shape of fatty acids, they can efficiently incorporate into lipid membranes and disturb membrane integrity, chain packing, and lateral pressure profile. These phenomena affect both model membranes as well as biological membranes. We investigated the feasibility of exploiting fatty acids as permeability enhancers in drug delivery systems for enhancing drug release from liposomal carriers and drug uptake by target cells. Saturated fatty acids, with acyl chain length from C8 to C20, were tested using model drug delivery liposomes of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the breast cancer MCF-7 cell line as a model cell. A calcein release assay demonstrated reduction in the membrane permeability barrier of the DPPC liposomes, proportionally to the length of the fatty acid. Differential scanning calorimetry (DSC) and dynamic light scattering (DLS) experiments revealed that C12 to C20 fatty acids can stabilize DPPC liposomal bilayers and induce the formation of large structures, probably due to liposome aggregation and bilayer morphological changes. On the other hand, the short fatty acids C8 and C10 tend to destabilize the bilayers and only moderately cause the formation of large structures. The effect of fatty acids on DPPC liposomes was not completely transferrable to the MCF-7 cell line. Using cytotoxicity assays, the cells were found to be relatively insensitive to the fatty acids at apoptotic sub-millimolar concentrations. Increasing the fatty acid concentration to few millimolar substantially reduced the viability of the cells, most likely via the induction of necrosis and cell lysis. A bioluminescence living-cell-based luciferase assay showed that saturated fatty acids in sub-cytotoxic concentrations cannot reduce the permeability barrier of cell membranes. Our results confirm that the membrane perturbing effect of fatty acids on model membranes cannot simply be carried over to biological membranes of live cells.

Immunomodulatory effects of bone marrow versus adipose tissue-derived mesenchymal stromal cells on NK cells: implications in the transplantation setting.

The ability of mesenchymal stromal cells (MSC) to suppress T-cell function has prompted their therapeutic use for graft-versus-host disease (GVHD) control. However, as MSC also modulate the activity of NK cells, which play an important role in graft-versus-leukemia (GVL) reaction, their administration could hamper this beneficial effect of allogeneic hematopoietic stem cell transplantation. MSC can be expanded from several sources, especially bone marrow and fat, but it is not well established if the cell source makes a difference in their immunoregulatory capacity. The aim of this study was to compare the immunomodulatory effect of MSC derived from bone marrow (BM-CSM) or adipose tissue (AT-MSC) on NK cells, to determine whether the use of MSC from one or the other origin could be more favorable to preserve NK cell activity and, therefore, GVL. Human NK cells were stimulated with IL-15 in the presence of BM-MSC or AT-MSC. The effect of both MSC populations on NK cell proliferation, cell cycle progression, and CD56 expression was analyzed by flow cytometry. Cytokine secretion was measured by ELISA, and cytotoxic activity was assessed by calcein release assays. Although both BM-MSC and AT-MSC induced a similar inhibition of NK cell proliferation, only BM-MSC decreased significantly NK cell cytotoxic activity and showed a trend for a higher reduction of IFN-γ secretion. These results suggest that, in the context of GVHD inhibition, the use of AT-MSC rather than BM-MSC could further preserve NK cell activity and, thus, favor GVL.

NKp46 expression identifies canine CD3 negative lymphocytes as natural killer cells with phenotypic and functional similarity to those in humans.

Abstract Canines are a unique model to study adoptive immunotherapy for cancer as they spontaneously develop many cancers with homology to human disease. However canine natural killer (NK) cells are poorly defined, rendering the model less useful for study of innate immune responses. NKp46 (NCR1) is an activating receptor expressed on NK cells and is considered a species-wide marker for NK cells, but its’ expression profile has not been demonstrated in canine. Therefore, we set out to determine whether NKp46 defines canine NK cells phenotypically and functionally. To this end, we generated a monoclonal mouse anti-canine NKp46 antibody by immunizing mice with L-cells expressing a canine NKp46 fusion protein. Canine peripheral blood cells were cultured in vitro with feeder cells expressing membrane bound human IL-21. The resultant cells were assessed for expression of NKp46, T-cell, B-cell, and macrophage markers. Expression of other NK cell genes were analyzed by RT-PCR. Cytotoxicity assays were conducted using a calcein-release assay with cancer cell lines. NKp46 is expressed on CD3− primary and cultured cells. Cultured CD3−/NKp46+ canine lymphocytes displayed spontaneous cytotoxicity against canine tumors and expressed NK cell activating receptors. CD3−/NKp46− (null) cultured cells were evident and had intermediate cytotoxicity between CD3+ and CD3−/NKp46+ cells. NKp46 MFI was inducible in these null cells following activation. We are currently investigating the cytokine, chemokine, and transcription factor profiling of CD3−/NKp46+ cells. We conclude that canine CD3−/NKp46+ cells are NK cells with phenotypic and functional similarity to human NK cells. In addition, canines have peripheral blood NK cell precursors that lack NKp46.

P2X receptor inhibition protects THP‐1 monocytes against cytolysis by α‐hemolysin from Escherichia Coli and leukotoxin from Aggregatibacter actinomycetemcomitans

The Repeats in Toxins (RTX) α‐hemolysin (HlyA) from E. coli and leukotoxin A (LtxA) from A. actinomycetemcomitans are important virulence factors for ascending urinary tract infections and aggressive forms of periodontitis respectively. Upon insertion into the plasma membrane ATP is released through the toxin pore and is as signaling molecule essential for the HlyA‐ and LtxA‐induced erythrocyte damage. The immediate effects after ATP release are a massive erythrocyte shrinkage and phosphatidylserine exposure in the outer leaflet of the plasma membrane. These events are eminent for the erythrocytes to be recognized and engulfed by the monocyte cell line THP‐1. Here, we investigate how these toxins and ATP, affect the survival and function of THP‐1 monocytes.By measurements of [Ca2+]i, we demonstrate that the THP‐1 express functional P2X (P2X1, P2X4 and P2X7) and P2Y2 receptor. We show that desensitization of either P2X1 by either low (0.001 μM) or more pronounced of P2X4/P2X7 by high concentrations of ATP (0.1 to 1 mM) increases the survival rate of THP‐1 cells attacked by either HlyA or LtxA in a calcein release assay. This protection is also found by specific blockage of the ATP sensitive receptors P2X1 (100 μM NF449) or P2X7 (100 μM oxATP or A804589). By erythrocyte phagocytosis assay, we determined that low dose HlyA (EC25) preconditioning is increasing the function of erythrocyte clearance at the place of injury.These findings suggest a new target for protecting monocytes during pore‐former toxemia – both pharmacologically through P2X blockers or receptor desenzitisation. Both mechanisms may be relevant for improving monocyte survival and function in patients suffering from infection with RTX producing bacteria.Support or Funding InformationFunded by the Danish Council for Independent Research, MEMRANES and Lundbeck Foundation

A Comparative Study on Techniques for the Determination of Permeabilization Events on Charged and Uncharged Lipid Bilayers

Host defense peptides (HDPs) are small effector molecules of the innate immune system building up the first defense barrier when it comes to infections. Infectious diseases are commonly treated with antibiotics leading to an increase in antibiotic resistances among clinically relevant pathogens. Therefore, understanding the mode of function of alternative agents with antibiotic function is undeniable for the successful development of new antibiotics opening new treatment options. To understand recognition-, recruiting- and permeabilization-processes on membrane surfaces a set of structurally diverse peptides is investigated regarding their membrane active function on charged and uncharged model membranes. Lipid bilayers are reconstituted from pure DOPC to mimic uncharged membranes and from POPE/POPG in a ratio of 4:1 to mimic the inner leaflet of the bactericidal cell envelope with an overall negative surface charge. Permeabilization events induced by the antimicrobial peptides LL32, Arenicin-1 and hBD3-l as well as the ionophore Nonactin and α-Hemolysin of Staphylococcus aureus are investigated with respect to the membrane systems stated above, buffer composition (high-salt vs. low-salt) and temperature conditions. A combination of a complex array of biophysical techniques is taken into account for studying permeabilization events with nearly no limitations - MONTAL-MUELLER-Setup for planar lipid bilayers, tethered membrane technology for supported bilayers, liposome-based assays (Calcein release assay, potassium iodide quenching, FRET-intercalation assay) and a chip-based pore spanning dye release assay. These methods allow to investigate the whole spectrum ranging from single pore characterization to the statistical analysis of either large membrane areas or a huge number of single membranes.

Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry Method.

Although natural killer (NK) cells produce various cytokines that regulate other lymphocytes of the immune system, the primary effector function of NK cells is the direct cytolysis of their targets. Hence analyzing the cytotoxic potential of these lymphocytes is fundamental to understanding their biology and their clinical impact. We have previously shown that release-based cytotoxicity assays, such as calcein release assay, could potentially underestimate percent specific lysis if the entrapped reporter is not completely released and demonstrated that an Image cytometry method can overcome this caveat. In this chapter, we describe a detailed methodology to quantitate NK cell cytotoxicity using the Cellometer Vision Image Cytometry system.

Membrane Disintegration Caused by the Steroid Saponin Digitonin Is Related to the Presence of Cholesterol.

In the present investigation we studied the molecular mechanisms of the monodesmosidic saponin digitonin on natural and artificial membranes. We measured the hemolytic activity of digitonin on red blood cells (RBCs). Also different lipid membrane models (large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs) in the presence and absence of cholesterol were employed. The stability and permeability of the different vesicle systems were studied by using calcein release assay, GUVs membrane permeability assay using confocal microscopy (CM) and fluorescence correlation spectroscopy (FCS) and vesicle size measurement by dynamic light scattering (DLS). The results support the essential role of cholesterol in explaining how digitonin can disintegrate biological and artificial membranes. Digitonin induces membrane permeability or causes membrane rupturing only in the presence of cholesterol in an all-or-none mechanism. This effect depends on the concentrations of both digitonin and cholesterol. At low concentrations, digitonin induces membrane permeability while keeping the membrane intact. When digitonin is combined with other drugs, a synergistic potentiation can be observed because it facilitates their uptake.

A Novel Method for Assessment of Natural Killer Cell Cytotoxicity Using Image Cytometry

Natural killer (NK) cells belong to the innate arm of the immune system and though activated NK cells can modulate immune responses through the secretion of cytokines, their primary effector function is through target cell lysis. Accordingly, cytotoxicity assays are central to studying NK cell function. The 51Chromium release assay, is the “gold standard” for cytotoxicity assay, however, due to concerns over toxicity associated with the use and disposal of radioactive compounds there is a significant interest in non-radioactive methods. We have previously used the calcein release assay as a non-radioactive alternative for studying NK cell cytotoxicity. In this study, we show that the calcein release assay varies in its dynamic range for different tumor targets, and that the entrapped calcein could remain unreleased within apoptotic bodies of lysed tumor targets or incompletely released resulting in underestimation of percent specific lysis. To overcome these limitations, we developed a novel cytotoxicity assay using the Cellometer Vision Image Cytometer and compared this method to standard calcein release assay for measuring NK cell cytotoxicity. Using tumor lines K562, 721.221, and Jurkat, we demonstrate here that image cytometry shows significantly higher percent specific lysis of the target cells compared to the standard calcein release assay within the same experimental setup. Image cytometry is able to accurately analyze live target cells by excluding dimmer cells and smaller apoptotic bodies from viable target cell counts. The image cytometry-based cytotoxicity assay is a simple, direct and sensitive method and is an appealing option for routine cytotoxicity assay.

Abstract 1303: Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells (effector), they releases the entrapped labels into the media upon lysis. The amount of labels in the media is measured to determine the level of cytotoxicity the effectors have induced. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel cytotoxicity assay using the Celigo imaging cytometry method. Utilizing imaging cytometry, direct cell counting of live fluorescent target cells can be performed, which is a direct method for assessment of cytotoxicity. Human NK cells from one healthy donor were used as effectors, and K562 (suspension) and IMR32 (adherent) were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at 10,000 cells/well in a standard 96-well microplate. The donor NK cells were then added to each well at Effector-to-Target (E:T) ratios 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. The 96 well plate was then scanned and analyzed using Celigo imaging cytometer at t = 1, 2, 3, and 4 h to measure the% lysis of target cells. The results showed increasing% lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity. Citation Format: Leo Li-Ying Chan, Kelsey Rosbach, Srinivas S. Somanchi, Dean Lee. Quantification of natural killer cell-mediated cytotoxicity using Celigo imaging cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1303. doi:10.1158/1538-7445.AM2015-1303

Heme Stabilization of α-Synuclein Oligomers during Amyloid Fibril Formation.

α-Synuclein (αSyn), which forms amyloid fibrils, is linked to the neuronal pathology of Parkinson's disease, as it is the major fibrillar component of Lewy bodies, the inclusions that are characteristic of the disease. Oligomeric structures, common to many neurodegenerative disease-related proteins, may in fact be the primary toxic species, while the amyloid fibrils exist either as a less toxic dead-end species or even as a beneficial mechanism for clearing damaged proteins. To alter the progression of the aggregation and gain insights into the prefibrillar structures, we determined the effect of heme on αSyn oligomerization by several different techniques, including native (nondenaturing) polyacrylamide gel electrophoresis, thioflavin T fluorescence, transmission electron microscopy, atomic force microscopy, circular dichroism, and membrane permeation using a calcein release assay. During aggregation, heme is able to bind the αSyn in a specific fashion, stabilizing distinct oligomeric conformations and promoting the formation of αSyn into annular structures, thereby delaying and/or inhibiting the fibrillation process. These results indicate that heme may play a regulatory role in the progression of Parkinson's disease; in addition, they provide insights into how the aggregation process may be altered, which may be applicable to the understanding of many neurodegenerative diseases.

Oligomers of Parkinson's Disease-Related α-Synuclein Mutants Have Similar Structures but Distinctive Membrane Permeabilization Properties.

Single-amino acid mutations in the human α-synuclein (αS) protein are related to early onset Parkinson's disease (PD). In addition to the well-known A30P, A53T, and E46K mutants, recently a number of new familial disease-related αS mutations have been discovered. How these mutations affect the putative physiological function of αS and the disease pathology is still unknown. Here we focus on the H50Q and G51D familial mutants and show that like wild-type αS, H50Q and G51D monomers bind to negatively charged membranes, form soluble partially folded oligomers with an aggregation number of ~30 monomers under specific conditions, and can aggregate into amyloid fibrils. We systematically studied the ability of these isolated oligomers to permeabilize membranes composed of anionic phospholipids (DOPG) and membranes mimicking the mitochondrial phospholipid composition (CL:POPE:POPC) using a calcein release assay. Small-angle X-ray scattering studies of isolated oligomers show that oligomers formed from wild-type αS and the A30P, E46K, H50Q, G51D, and A53T disease-related mutants are composed of a similar number of monomers. However, although the binding affinity of the monomeric protein and the aggregation number of the oligomers formed under our specific protocol are comparable for wild-type αS and H50Q and G51D αS, G51D oligomers cannot disrupt negatively charged and physiologically relevant model membranes. Replacement of the membrane-immersed glycine with a negatively charged aspartic acid at position 51 apparently abrogates membrane destabilization, whereas a mutation in the proximal but solvent-exposed part of the membrane-bound α-helix such as that found in the H50Q mutant has little effect on the bilayer disrupting properties of oligomers.

High-throughput direct cell counting-based natural killer cell-mediated cytotoxicity assay using Celigo Imaging Cytometry (TUM2P.1027)

Abstract Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells, they releases the entrapped labels into the media upon lysis. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel high-throughput direct cell counting method for cytotoxicity assay using the Celigo imaging cytometry method. Human NK cells from one healthy donor were used as effectors, and K562 and IMR32 were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at in a 96-well microplate. NK cells were then added to each well at various Effector-to-Target (E:T) ratios. The 96 well plate was then scanned and analyzed using the Celigo to measure the % lysis of target cells at different time points. The results showed increasing % lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.

Construction of Novel Thermosensitive Magnetic Cationic Liposomes as a Drug and Gene Co-Delivery System.

This study aimed to develop a novel system for co-delivery of both drugs and genes to the same cells. We described the construction of thermosensitive magnetic cationic liposomes (TSMCLs). Liposomes were prepared by thin film hydration method, then calcein release assay and DSC were used to determine the thermosensitivity of liposomes, and gel retardation experiment was performed to monitor the formation of Lipoplex (Liposomes and pDNA complex), finally the in vitro transfection experiments were performed to evaluate the transfection efficiency of the liposomes. The results showed that TSMCLs combined the features of physical targeting under magnetic guidance and hyperthermia triggered drug release upon the application of alternating magnetic field. Thus TSMCLs could be used for the co-delivery of drugs and genes and have potential application in combined chemotherapy and gene therapy for cancer.

Comparison of Ibrutinib, Idelalisib and Olaptesed Pegol on the Immune Effector Function Mediated By Rituximab

Olaptesed pegol (NOX-A12), an SDF-1 binding Spiegelmer® was found to detach SDF-1 from the surface of BMSCs (Hoellenriegel et al., Blood 2013) and to mobilize CXCR4 expressing, malignant cells from protective niches in the bone marrow or other secondary lymphoid tissues, thereby sensitizing them to the action of standard therapy. In addition to malignant cells CXCR4 expressing immune cells, e.g. T cells, neutrophils and monocytes are effectively mobilized by olaptesed (Vater et al., Clin Pharmacol Ther 2013). Here we investigated the combination of olaptesed with an immunotherapeutic agent, in particular rituximab (RTX). Several mechanisms of action (MoA) of RTX have been described including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). The aim was to analyze whether olaptesed influences the immune effector cell activation by RTX as has been shown for the Btk or PI3Kd inhibitors ibrutinib and idelalisib, respectively.ADCC activity was measured by the calcein release assay. Raji lymphoma cells were labeled with calcein, treated with 10 µg/mL RTX and co-cultured for 4 hours with various ratios of PBMCs as effector cells, which have been incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, overnight. In order to analyze the impact on phagocyte function, ADCP assays with neutrophils and monocytes were performed. Phagocytes were preincubated with either 10 µM olaptesed, 1 µM ibrutinib or 1 µM idelalisib, respectively, for 6 hours, and cocultured overnight with DiD-labeled primary B-cells or Raji cells which were opsonized with different concentrations of RTX. ADCP was quantified in a guava easyCyte flow cytometer by counting the CD15+ (or CD14+) and DiD+ population (gated on CD19-negative cells) and normalizing to total phagocyte counts. To analyze B-cell depletion, heparinized whole blood from healthy volunteers was diluted with HBSS (1:2.5) and incubated with either 10 µM olaptesed, 1 µM ibrutinib or 1µM idelalisib, respectively, for 6 hours followed by the addition of different RTX concentrations. After incubation for 16 hours B-cell depletion was evaluated by determining the CD19+ B-cell and CD3+ T-cell populations.We found that preincubation of PBMCs with 1 µM ibrutinib significantly decreased calcein release. 1 µM idelalisib led to a slight decrease of calcein release, while 10 µM olaptesed did not influence ADCC activity (Figure 1A). Preliminary results from ADCP assays point to a decreased phagocytic activity in terms of maximal ADCP for neutrophils after preincubation with ibrutinib or idelalisib (Figure 1B). Importantly, although olaptesed is being endocytosed by phagocytes, the uptake of the drug did not inhibit ADCP. In accordance, the whole blood assay showed that olaptesed had no negative impact on RTX-mediated B-cell depletion, while ibrutinib and idelalisib were found to decrease B-cell depletion regarding EC50 and maximal killing (Figure 1C). Taken together, we found that olaptesed retains the immune functional repertoire of RTX, whereas ibrutinib and idelalisib decrease the immune effector function. These small molecule kinase inhibitors possess low specificity and are inhibiting also other kinases which may be important for immune cell activation, such as ITK which is blocked by ibrutinib. The MoA of olaptesed is partially overlapping with kinase inhibitors with regard to lymphoid cell mobilization; however, olaptesed is highly selective and therefore does not impair immune-cell mediated contribution to monoclonal antibody efficacy. Instead, olaptesed may further enhance the contribution of immune effector cells through mobilization of T cells from the bone marrow which are described to be exhausted in the peripheral blood of hematological malignancies like CLL. Furthermore, neutrophils and monocytes which contribute to ADCP are effectively mobilized with olaptesed.Based on their complementary mechanisms of action involving direct and immune-mediated cell death (RTX) or long-term mobilization of not only the malignant cells but also effector immune cells (olaptesed), the combination has the potential for greater efficacy in treating B lymphoid malignancies without impairing immune effector activity. Olaptesed is currently being tested in a Phase 2a clinical trial in combination with RTX and bendamustine in patients with relapsed/refractory CLL (#NCT01486797). [Display omitted] DisclosuresZboralski:NOXXON Pharma AG: Employment. Vater:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.