Real-time Confocal Imaging Research Articles

Improving nuclear morphometry imaging with real-time and low-cost line-scanning confocal microendoscope.

Fiber-optic endomicroscopy is a minimally invasive tool to probe disease progression with subcellular resolution. In this Letter, we demonstrate a low-cost and compact fluorescence microendoscope capable of line-scanning confocal imaging by synchronizing a digital light projector with a CMOS camera. We present the digital aperture design to enable real-time confocal imaging, and we implement parallel illumination to improve the optical sectioning performance. Furthermore, we show that the confocal microendoscope can enhance visualization of disease-associated features when imaging highly scattering esophageal specimens.

Differential response of human dermal fibroblast subpopulations to visible and near-infrared light: Potential of photobiomodulation for addressing cutaneous conditions.

The past decade has witnessed a rapid expansion of photobiomodulation (PBM), demonstrating encouraging results for the treatment of cutaneous disorders. Confidence in this approach, however, is impaired not only by a lack of understanding of the light-triggered molecular cascades but also by the significant inconsistency in published experimental outcomes, design of the studies and applied optical parameters. This study aimed at characterizing the response of human dermal fibroblast subpopulations to visible and near-infrared (NIR) light in an attempt to identify the optical treatment parameters with high potential to address deficits in aging skin and non-healing chronic wounds. Primary human reticular and papillary dermal fibroblasts (DF) were isolated from the surplus of post-surgery human facial skin. An in-house developed LED-based device was used to irradiate cell cultures using six discrete wavelengths (450, 490, 550, 590, 650, and 850 nm). Light dose-response at a standard oxygen concentration (20%) at all six wavelengths was evaluated in terms of cell metabolic activity. This was followed by an analysis of the transcriptome and procollagen I production at a protein level, where cells were cultured in conditions closer to in vivo at 2% environmental oxygen and 2% serum. Furthermore, the production of reactive oxygen species (ROS) was accessed using real-time fluorescence confocal microscopy imaging. Here, production of ROS in the presence or absence of antioxidants, as well as the cellular localization of ROS, was evaluated. In terms of metabolic activity, consecutive irradiation with short-wavelength light (⇐530 nm) exerted an inhibitory effect on DF, while longer wavelengths (>=590 nm) had essentially a neutral effect. Cell behavior following treatment with 450 nm was biphasic with two distinct states: inhibitory at low- to mid- dose levels (<=30 J/cm2 ), and cytotoxic at higher dose levels (>30 J/cm2 ). Cell response to blue light was accompanied by a dose-dependent release of ROS that was localized in the perinuclear area close to mitochondria, which was attenuated by an antioxidant. Overall, reticular DFs exhibited a greater sensitivity to light treatment at the level of gene expression than did papillary DFs, with more genes significantly up- or down- regulated. At the intra-cellular signaling pathway level, the up- or down- regulation of vital pathways was observed only for reticular DF, after treatment with 30 J/cm2 of blue light. At the cellular level, short visible wavelengths exerted a greater inhibitory effect on reticular DF. Several genes involved in the TGF-β signaling pathway were also affected. In addition, procollagen I production was inhibited. By contrast, 850 nm near-infrared (NIR) light (20 J/cm2 ) exerted a stimulatory metabolic effect in these cells, with no detectable intracellular ROS formation. Here too, reticular DF were more responsive than papillary DF. This stimulatory effect was only observed under in vivo-like low oxygen conditions, corresponding to normal dermal tissue oxygen levels (approximately 2%). This study highlights a differential impact of light on human skin cells with upregulation of metabolic activity with NIR light, and inhibition of pro-collagen production and proliferation in response to blue light. These findings open-up new avenues for developing therapies for different cutaneous conditions (e.g., treatment of keloids and fibrosis) or differential therapy at distinct stages of wound healing. Lasers Surg. Med. 50:859-882, 2018. © 2018 Wiley Periodicals, Inc.

Abstract LB-220: A microfluidic model for real-time monitoring of tumor-lymphocyte interactions

Abstract The emergence of immune checkpoint inhibitors and the promise of increased patient survival reinforces the potential for targeting the tumor microenvironment as a drug development strategy. The tumor microenvironment consists of a heterogeneous composition of cancer cells, vascular cells, fibroblasts, and immune cells that are regulated by both intrinsic and extrinsic chemical and physical stimuli. Within this microenvironment, aberrant blood vessel formation and extra cellular matrix (ECM) restructuring lead to increased interstitial fluid flow and shear stress that stimulate differential gene expression, reorganization of tumor cell structure, and suppression of local cytotoxic T-cell activity. Elevated levels of fluid pressure and shear stress have also been demonstrated to alter cell sensitivity and response to therapeutic agents. Traditional static 2D in vitro models fail to adequately address tumor-immune cell interactions and flow mediated change in sensitivity, contributing to inaccurate predictions of in vivo therapeutic response. The development of 3D microfluidic systems have greatly enhanced the ability to model components of the tumor microenvironment, such as fluid flow, and capture the dynamics of tumor and immune cell responses to emerging therapeutic compounds. Here, we have developed a medium throughput microfluidic device which is constructed using bioinert materials that are also compatible with real-time confocal imaging, to model the effects of therapeutic treatment on tumor viability. In addition to exposing tumors to a dynamic flow environment, this model system enables the introduction of flowing lymphocytes, permitting tracking of tumor-lymphocyte interactions in real-time. As a test case, we demonstrate the ex vivo binding and cytotoxic activity of MC38-derived tumor infiltrating lymphocytes (TILs) on tumor fragments. These results highlight the power of a dynamic microfluidic flow system to model the tumor-lymphocyte microenvironment and the potential of this system to more accurately evaluate novel therapeutic agents. Citation Format: Nathan Moore, Daniel Doty, Jose Santos, Louis B. Kratchman, Stephanie Angione, Vienna Mott, Mark Zielstroff, Hongmin Chen, Alla Gimbel, Jeffrey Borenstein. A microfluidic model for real-time monitoring of tumor-lymphocyte interactions [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 LB-220. doi:10.1158/1538-7445.AM2017-LB-220

A Nanoparticle-Decorated Biomolecule-Responsive Polymer Enables Robust Signaling Cascade for Biosensing.

To meet the increasing demands for ultrasensitivity in monitoring trace amounts of low-abundance early biomarkers or environmental toxins, the development of a robust sensing system is urgently needed. Here, a novel signal cascade strategy is reported via an ultrasensitive polymeric sensing system (UPSS) composed of gold nanoparticle (gNP)-decorated polymer, which enables gNP aggregation in polymeric network and electrical conductance change upon specific aptamer-based biomolecular recognition. Ultralow concentrations of thrombin (10-18 m) as well as a low molecular weight anatoxin (165 Da, 10-14 m) are detected selectively and reproducibly. The biomolecular recognition induced polymeric network shrinkage responses as well as dose-dependent responses of the UPSS are validated using in situ real-time atomic-force microscopy, representing the first instance of real-time detection of biomolecular binding-induced polymer shrinkage in soft matter. Furthermore, in situ real-time confocal laser scanning microscopy imaging reveals the dynamic process of gNP aggregation responses upon biomolecular binding.

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review.

BackgroundMultiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing.MethodsIn vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging.ResultsPMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis.ConclusionOur systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method.

We demonstrated GPU accelerated real-time confocal fluorescence lifetime imaging microscopy (FLIM) based on the analog mean-delay (AMD) method. Our algorithm was verified for various fluorescence lifetimes and photon numbers. The GPU processing time was faster than the physical scanning time for images up to 800 × 800, and more than 149 times faster than a single core CPU. The frame rate of our system was demonstrated to be 13 fps for a 200 × 200 pixel image when observing maize vascular tissue. This system can be utilized for observing dynamic biological reactions, medical diagnosis, and real-time industrial inspection.

"Race for the Surface": Eukaryotic Cells Can Win.

With an aging population and the consequent increasing use of medical implants, managing the possible infections arising from implant surgery remains a global challenge. Here, we demonstrate for the first time that a precise nanotopology provides an effective intervention in bacterial cocolonization enabling the proliferation of eukaryotic cells on a substratum surface, preinfected by both live Gram-negative, Pseudomonas aeruginosa, and Gram-positive, Staphylococcus aureus, pathogenic bacteria. The topology of the model black silicon (bSi) substratum not only favors the proliferation of eukaryotic cells but is biocompatible, not triggering an inflammatory response in the host. The attachment behavior and development of filopodia when COS-7 fibroblast cells are placed in contact with the bSi surface are demonstrated in the dynamic study, which is based on the use of real-time sequential confocal imaging. Bactericidal nanotopology may enhance the prospect for further development of inherently responsive antibacterial nanomaterials for bionic applications such as prosthetics and implants.

Sonoprinting and the importance of microbubble loading for the ultrasound mediated cellular delivery of nanoparticles

In the last years, research on ultrasound mediated drug delivery using microbubbles is vastly expanding. While some groups simply mix drugs and microbubbles (co-administration), other researchers have a major interest in the potential of drug-loaded microbubbles. However, today, little is known on the pros and cons of these two strategies. In this study we evaluated the delivery of nanoparticles (polystyrene nanospheres and mRNA-lipoplexes) to cells in vitro, in case the nanoparticles were mixed with unloaded microbubbles versus loaded onto the microbubbles. Flow cytometry experiments demonstrated that unloaded microbubbles did not enhance the cellular delivery of the nanospheres and mRNA-lipoplexes. However, upon loading the nanoparticles onto the microbubbles, their delivery to cells substantially improved. Real-time swept field confocal microscopy imaging of the microbubbles and cells during ultrasound radiation revealed that nanoparticle-loaded microbubbles directly deposited the nanoparticles in patches onto the cell membrane, a process that we termed ‘sonoprinting’. This phenomenon resulted in the delivery of large amounts of nanoparticles to the cells and is suggested to be different from the creation of cell membrane pores and enhanced endocytosis, which have been reported before as mechanisms behind the improved delivery of drugs to cells by ultrasound.

Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions.

New Insights into the Prothrombotic State of Heparin-Induced Thrombocytopenia (HIT) Using a Novel Microfluidic System: The Endothelial Lining and “Rolling Barrage” of Activation

The most feared feature of HIT is antibody-mediated thrombosis. We have shown that this prothrombotic state is related to binding of platelet factor 4 (PF4), a chemokine densely packaged into platelet alpha-granules, to surface glycosaminoglycans (GAGs) expressed on hematopoietic and vascular cells. PF4/GAG surface complexes are recognized by HIT antibodies, activating the targeted cells. Unlike platelets that express only low-affinity chondroitin sulfate surface GAGs, endothelial cells (EC) express a glycocalyx enriched in heparan sulfate, which has higher affinity for PF4, potentially increasing their propensity to become a target for immune injury leading to thrombosis. We examined the details of the development of in situ thrombi using the cremaster arteriole laser injury model beginning with transgenic mice expressing only human PF4 (hPF4+), but lacking FcγRIIA. These mice do not develop thrombocytopenia or thrombosis when injected with the HIT-like monoclonal antibody KKO or IgGs isolated from patients with HIT. In these mice, antigenic PF4/GAG complexes were recognized by KKO at sites of vascular injury even in the absence of infused heparin. In fact, infusion of sufficient heparin dissociated PF4 from sites of injury, consistent with its higher affinity for PF4 than cell surface GAGs. This suggests that antigenic PF4/GAG complexes normally develop intravascularly whenever thrombus formation occurs, yet these complexes do not typically initiate antibody-mediated thrombosis. Real-time confocal imaging of injured vessels revealed that PF4 first bound almost exclusively to the peri-injury endothelium. This was especially evident immediately upstream of the thrombus where turbulent blood flow may lead to platelet degranulation and subsequent adherence of the released PF4 to the glycocalyx. Beginning approximately two minutes post-injury, binding of KKO, presumably to PF4/GAG complexes on platelets, is seen at the interface between the shell and core of the thrombus. We then repeated these same studies in hPF4+/FcγRIIA+ mice, where infused KKO or HIT IgGs leads to significant thrombocytopenia and widespread development of thrombi as in HIT. Similar adherence of PF4 to the peri-injury EC and then to the core/shell interface of the thrombus as seen in the hPF4+ mice, but the changes were more extensive in hPF4+/FcγRIIA+ mice after KKO infusion and often lead to vascular occlusion. To further define the basis of the prothrombotic state in HIT and to extend our studies to a human system, we examined thrombus formation in HIT in a novel microfluidic system in which vascular injury was induced in an upstream portion of a human umbilical vein EC-lined channel by reactive oxygen species generated through excitation of infused hematoporphyrin by blue light (490 nm). Following infusion of human blood, platelets accumulated and released PF4, which bound the injured endothelium, while the downstream endothelium remained quiescent. Addition of KKO to the infused whole blood lead to a HIT-like state with marked increase in platelet adhesion and binding of PF4 to the injured endothelium, but binding of PF4 now spread downstream of the boundary between injured and uninjured endothelium. This was followed by downstream platelet adhesion and often occlusion of the channel. We proposed that this spread in EC injury was a result of a “rolling barrage” of PF4 released from platelets binding to the injured patch of EC complexing to the downstream glycocalyx on the non-injured endothelium followed by KKO binding and subsequent endothelial activation. The newly activated ECs bound additional platelets and the process repeats, rolling downstream and extending thrombus growth. Thus, these studies provide important new insights into the local sequence of events that propagate clots in HIT: Targeting of the endothelial glycocalyx by HIT antibodies is a major contributor to the prothrombotic state. Platelets adherent to the site of original injury release PF4, which then binds to downstream EC glycocalyx and initiates repetitive cycles of PF4 binding, EC activation and platelet adherence, and further release of PF4 that propagates growth of thrombi to previously uninvolved vasculature. DisclosuresNo relevant conflicts of interest to declare.

Digitally synthesized beat frequency-multiplexed fluorescence lifetime spectroscopy.

Frequency domain fluorescence lifetime imaging is a powerful technique that enables the observation of subtle changes in the molecular environment of a fluorescent probe. This technique works by measuring the phase delay between the optical emission and excitation of fluorophores as a function of modulation frequency. However, high-resolution measurements are time consuming, as the excitation modulation frequency must be swept, and faster low-resolution measurements at a single frequency are prone to large errors. Here, we present a low cost optical system for applications in real-time confocal lifetime imaging, which measures the phase vs. frequency spectrum without sweeping. Deemed Lifetime Imaging using Frequency-multiplexed Excitation (LIFE), this technique uses a digitally-synthesized radio frequency comb to drive an acousto-optic deflector, operated in a cat's-eye configuration, to produce a single laser excitation beam modulated at multiple beat frequencies. We demonstrate simultaneous fluorescence lifetime measurements at 10 frequencies over a bandwidth of 48 MHz, enabling high speed frequency domain lifetime analysis of single- and multi-component sample mixtures.

Hydrogen sulfide upregulates KATP channel expression in vascular smooth muscle cells of spontaneously hypertensive rats.

The study was designed to investigate whether H2S could upregulate expression of KATP channels in vascular smooth muscle cells (VSMCs), and by this mechanism enhances vasorelaxation in spontaneously hypertensive rats (SHR). Blood pressure, vascular structure, and vasorelaxation were analyzed. Plasma H2S was detected using polarographic sensor. SUR2B and Kir6.1 expressions were detected in VSMCs of SHR and in A7r5 cells as well as primarily cultured ASMCs using real-time PCR, western blot, immunofluorescence, and confocal imaging. Nuclear translocation of forkhead transcription factors FOXO1 and FOXO3a in ASMCs was detected using laser confocal microscopy, and their binding activity with SUR2B and Kir6.1 promoters was examined by chromatin immunoprecipitation. SHR developed hypertension at 18weeks. They showed downregulated vascular SUR2B and Kir6.1 expressions in association with a decreased plasma H2S level. H2S donor, however, could upregulate vascular SUR2B and Kir6.1 expressions, causing a left shift of the vasorelaxation curve to pinacidil and lowered tail artery pressure in the SHR. Also, H2S antagonized endothelin-1 (ET-1)-inhibited KATP expression in A7r5 cells and cultured ASMCs. Mechanistically, H2S inhibited ET-1-stimulated p-FOXO1 and p-FOXO3a expressions (inactivated forms), but increased their nuclear translocation and the ET-1-inhibited binding of FOXO1 and FOXO3a with Kir6.1 and SUR2B promoters in ASMCs. Hence, H2S promotes vasorelaxation of SHR, at least in part, through upregulating the expression of KATP subunits by inhibiting phosphorylation of FOXO1 and FOXO3a, and stimulating FOXO1 and FOXO3a nuclear translocation and their binding activity with SUR2B and Kir6.1 promoters. H2S increased vascular SUR2B and Kir6.1 expression of SHR, promoting vasorelaxation. H2S antagonized ET-1-inhibited KATP expression in A7r5 cells and cultured ASMCs. H2S inhibited ET-1-induced FOXO1 and FOXO3a phosphorylation in ASMCs. H2S promoted FOXO1 and FOXO3a nuclear translocation and binding with target gene promoters.

531Platelet-derived deoxyribose-1-phosphate promotes endothelial cell motility and angiogenesis in vivo via redox stimulation

Redox regulation of angiogenesis is an important and emerging topic in vascular sciences; reactive oxygen species (ROS) modulate vascular endothelial cell (EC) proliferation and migration, but the molecular mechanism underlying this phenomenon remains largely unclear. We have shown micromolar concentrations of the pro-angiogenic metabolite deoxyribose-1-phosphate (dRP) detected by mass spectrometry (MS) and nuclear magnetic resonance (NMR) in the supernatants of collagen- and thrombin-stimulated platelets. The genetic silencing of dRP-generating enzymes uridine phosphorylase (UP) and thymidine phosphorylase (TP) in mouse platelets significantly reduced the levels of dRP in platelet supernatants and impaired the ability of protein-free platelet supernatants to enhance human umbilical vein endothelial cell (HUVEC) motility. Chick chorioallantoic membrane (CAM) vascularisation assays performed with protein-free supernatants suggested that the release of dRP by platelets stimulates angiogenesis in vivo. To understand the pro-angiogenic effect of dRP, we investigated further the molecular mechanism of action of dRP and whether this effect was observed in other nucleoside derivatives deoxyribose-5-phospate (dR5P), ribose-1-phosphate (R1P) and deoxyribose (dR). Using live cell real-time confocal imaging and fluorescence assays, HUVEC treatment with dRP significantly increased ROS generation. Similarly, dR5P and R1P also induced ROS accumulation in HUVECs, whereas dR did not. The protein expression of the redox stress marker heme oxygenase-1 (HO-1) and Integrin β3 were shown to be upregulated by dRP and its phosphorylated derivatives (except dR) by immunoblot experiments. Vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), interleukin 8 (IL-8), and stromal cell-derived factor 1α (SDF-1α) showed a similar effect. In parallel, only dRP and its phosphorylated derivatives increased HUVEC cell motility in scratch-healing assays and capillary tube formation using Matrigel™ assays. The causative link between integrin β3 upregulation and increased HUVEC motility/capillary tube formation was proved using integrin-specific inhibitory antibodies'. Furthermore the angiogenic potential of dRP was confirmed in vivo. In summary, dRP and its phosphorylated derivatives were shown to play a relevant role in the paracrine regulation of EC motility and pro-angiogenic activity by up-regulating integrin β3 in a ROS-dependent manner possibly signaling via VEGFR2. dRP is likely to play a role in the stimulation of postnatal angiogenesis and tissue regeneration.

Confocal Imaging with a Fluorescent Bile Acid Analogue Closely Mimicking Hepatic Taurocholate Disposition

This study aimed to characterize the in vitro hepatic transport mechanisms in primary rat and human hepatocytes of the fluorescent bile acid derivative N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD), previously synthesized to study the activity of the bile salt export pump (BSEP). The fluorescent bile acid derivative exhibited saturable uptake kinetics in suspended rat hepatocytes. Hepatic uptake was inhibited in the presence of substrates/inhibitors of the organic anion transporting polypeptide (Oatp) family and Na(+) -taurocholate cotransporting peptide (Ntcp). Concentration-dependent uptake of the fluorescent bile acid was also saturable in Chinese hamster ovary cells transfected with rNtcp, hNTCP, OATP1B1, or OATP1B3. The fluorescent bile acid derivative was actively excreted in the bile canaliculi of sandwich-cultured rat and human hepatocytes (SCRH and SCHH), with a biliary excretion index (BEI) of 26% and 32%, respectively. In SCRH, cyclosporin A significantly decreased the BEI to 5%. Quantification by real-time confocal imaging further confirmed canalicular transport of the fluorescent bile acid derivative (BEI = 75%). We conclude that tauro-nor-THCA-24-DBD is a useful probe to study interference of drugs with NTCP/Ntcp- and BSEP/Bsep-mediated transport in fluorescence-based in vitro assays.

Real-time evaluation of aggregation using confocal imaging and image analysis tools

Real-time confocal imaging was utilised to monitor the in situ loss of BSA monomers and aggregate formation using Spatial Intensity Distribution Analysis (SpIDA) and Raster Image Correlation Spectroscopy (RICS). At the proof of concept level this work has demonstrated the applicability of RICS and SpIDA for monitoring protein oligomerisation and larger aggregate formation.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or &lt;em&gt;In Vivo&lt;/em&gt;

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Abstract A83: Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy.

Abstract Antitumor therapy with monoclonal antibodies has shown great therapeutic benefits in clinics and a number of therapeutic antibodies have been approved for use in patients. Nevertheless, their efficacy is limited due to their insufficient inherent cell killing activity. In order to augment their antitumor efficacy, monoclonal antibodies may be modified with natural toxins to create a drug conjugate (immunotoxin) that comprises the dual functionality of monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) and the cell-killing activity of the toxin. In our investigations, two immunotoxins were prepared by chemically coupling the monoclonal antibodies trastuzumab (Herceptin®) and cetuximab (Erbitux®) to the plant derived toxin saporin via a cleavable disulfide bond. Impedance-based real-time viability assays and confocal live cell imaging revealed that the toxin is efficiently delivered to the targeted tumor cells. In addition, immunotoxin cytotoxicity was tremendously enhanced in combination with certain plant glycosides, which specifically mediate the endo/lysosmal release of the toxin moiety into the cytosol without damaging the biological membranes. The combinatorial approach was successful in achieving a complete cell death of human breast ductal carcinoma BT-474 cells, at a concentration of 1 nM of trastuzumab-saporin or 0.001 nM cetuximab-saporin in the presence of the glycosides. Furthermore, immunotoxins retained their ability to interact with the innate immune system and trigger antibody-dependent cell-mediated cytotoxicity. The combinatorial approach is conceived as a novel and promising way to increase the efficacy of monoclonal antibodies in targeted tumor therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A83. Citation Format: Roger Gilabert-Oriol, Mayank Thakur, Benedicta von Mallinckrodt, Thomas Hug, Burkhard Wiesner, Jenny Eichhorst, Matthias F. Melzig, Hendrik Fuchs, Alexander Weng. Combinatorial approach to drastically enhance the monoclonal antibody efficacy in targeted tumor therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A83.

Modified Trastuzumab and Cetuximab Mediate Efficient Toxin Delivery While Retaining Antibody-Dependent Cell-Mediated Cytotoxicity in Target Cells

Monoclonal antibody-based therapy is one of the most successful strategies for treatment of cancer. However, the insufficient cell killing activity of monoclonal antibodies limits their therapeutic potential. These limitations can be overcome by the application of immunotoxins, which consist of a monoclonal antibody that specifically delivers a toxin into the cancer cell. An ideal immunotoxin combines the functionality of the monoclonal antibody (antagonistic binding to targeted receptors and interaction with the innate immune system) with the cell-killing activity of the toxic moiety. In addition, it should be sensitive for certain triterpenoid saponins that are known to lead to a tremendous augmentation of the antitumoral efficacy of the immunotoxin. In this study, the monoclonal antibodies trastuzumab (Herceptin) and cetuximab (Erbitux) were conjugated via cleavable disulfide bonds to the plant derived toxin saporin. The ability of the modified tumor-specific therapeutic antibodies to deliver their toxic payload into the target cells was investigated by impedance-based real-time viability assays and confocal live cell imaging. We further provide evidence that the immunotoxins retained their ability to trigger antibody-dependent cell-mediated cytotoxicity. They specifically bound to their target cell receptor, and their cell-killing activity was drastically augmented in the presence of triterpenoid saponins. Further mechanistic studies indicated a specific saponin-mediated endo/lysosomal release of the toxin moiety. These results open a promising avenue to overcome the present limitations of therapeutic antibodies and to achieve a higher antitumoral efficacy in cancer therapy.

Abstract 153: Angiotensin II-induced Hypertension Alters Endothelial CaMKII

The endothelial dysfunction associated with arterial hypertension is characterized by a Ca2+ dyshomeostasis resulting in an alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO). However, the underlying mechanisms remain controversial. It has been recently suggested that CaMKII, a Ca2+-calmodulin dependent kinase plays an important role in cardiovascular diseases such as diabetes-associated hypertension. Paradoxically, CaMKII also modulates eNOS expression and activity, and is thereby involved in the regulation of endothelial function. Indeed, acting as a Ca2+ sensor, CaMKII has the unique ability to integrate oscillatory Ca2+ signals into specific outcomes. We recently provided the first evidence that endothelial CaMKII is activated by a local Ca2+ signal, the Ca2+ pulsars, localized within myoendothelial projections (MEP), and results in an increased NO production. The characterization of this novel endothelial CaMKII signalling pathway in pathophysiological conditions will strengthen our understanding of endothelial dysfunction associated with hypertension. This study aimed at the characterization of the impact of AngII-induced hypertension on the correlation between endothelial Ca2+pulsars and CaMKII in microvasculature. Real-time confocal imaging showed that acute exposure to AngII (10 μM) increases both ROS intracellular levels (52%) and Ca2+ pulsars frequency (35%). Exposure to lower AngII levels (100 nM) only increased Ca2+ pulsars frequency (49%). However, CaMKIIα and β clustering (and activation) within MEP was observed at both AngII levels. Acute AngII also stimulated NO production (42%), an increase blunted by CaMKII inhibition with KN-93 (17%). Although a significant increase in Ca2+ pulsars frequency was observed (147%) in AngII-hypertensive mice, an AngII-induce increased in endothelial ROS levels was recorded independently of blood pressure levels. Recruitment of CaMKIIα and β at the MEP by chronic AngII was also increased regardless of blood pressure (60% and 43%). Our finding strongly suggests that AngII stimulates CaMKII through both ROS and Ca2+ pulsars . This study consolidates our understanding of CaMKII as a key regulatory component of endothelial function.

Eukaryotic Translation Initiation Factor 3 Subunit E Controls Intracellular Calcium Homeostasis by Regulation of Cav1.2 Surface Expression

Inappropriate surface expression of voltage-gated Ca2+channels (CaV) in pancreatic ß-cells may contribute to the development of type 2 diabetes. First, failure to increase intracellular Ca2+ concentrations at the sites of exocytosis impedes insulin release. Furthermore, excessive Ca2+ influx may trigger cytotoxic effects. The regulation of surface expression of CaV channels in the pancreatic β-cells remains unknown. Here, we used real-time 3D confocal and TIRFM imaging, immunocytochemistry, cellular fractionation, immunoprecipitation and electrophysiology to study trafficking of L-type CaV1.2 channels upon β-cell stimulation. We found decreased surface expression of CaV1.2 and a corresponding reduction in L-type whole-cell Ca2+ currents in insulin-secreting INS-1 832/13 cells upon protracted (15–30 min) stimulation. This internalization occurs by clathrin-dependent endocytosis and could be prevented by microtubule or dynamin inhibitors. eIF3e (Eukaryotic translation initiation factor 3 subunit E) is part of the protein translation initiation complex, but its effect on translation are modest and effects in ion channel trafficking have been suggested. The factor interacted with CaV1.2 and regulated CaV1.2 traffic bidirectionally. eIF3e silencing impaired CaV1.2 internalization, which resulted in an increased intracellular Ca2+ load upon stimulation. These findings provide a mechanism for regulation of L-type CaV channel surface expression with consequences for β-cell calcium homeostasis, which will affect pancreatic β-cell function and insulin production.