Intravital Confocal Microscopy Research Articles

CCN1/CYR61-mediated meticulous patrolling by Ly6Clow monocytes fuels vascular inflammation

Inflammation is characterized by the recruitment of leukocytes from the bloodstream. The rapid arrival of neutrophils is followed by a wave of inflammatory lymphocyte antigen 6 complex (Ly6C)-positive monocytes. In contrast Ly6C(low) monocytes survey the endothelium in the steady state, but their role in inflammation is still unclear. Here, using confocal intravital microscopy, we show that upon Toll-like receptor 7/8 (TLR7/8)-mediated inflammation of mesenteric veins, platelet activation drives the rapid mobilization of Ly6C(low) monocytes to the luminal side of the endothelium. After repeatedly interacting with platelets, Ly6C(low) monocytes commit to a meticulous patrolling of the endothelial wall and orchestrate the subsequent arrival and extravasation of neutrophils through the production of proinflammatory cytokines and chemokines. At a molecular level, we show that cysteine-rich protein 61 (CYR61)/CYR61 connective tissue growth factor nephroblastoma overexpressed 1 (CCN1) protein is released by activated platelets and enables the recruitment of Ly6C(low) monocytes upon vascular inflammation. In addition endothelium-bound CCN1 sustains the adequate patrolling of Ly6C(low) monocytes both in the steady state and under inflammatory conditions. Blocking CCN1 or platelets with specific antibodies impaired the early arrival of Ly6C(low) monocytes and abolished the recruitment of neutrophils. These results refine the leukocyte recruitment cascade model by introducing endothelium-bound CCN1 as an inflammation mediator and by demonstrating a role for platelets and patrolling Ly6C(low) monocytes in acute vascular inflammation.

Abstract PR14: Epidermal neoplasms bypass immune evasion requirements via niche occupancy

Abstract Anti-tumor immune effectors exert selective pressure against transformed clones, ultimately permitting only mutation cascades that are either non-immunogenic or include immune-evasive adaptations. This evolutionary process often includes an equilibrium phase during which immunogenic transformed cells are prevented from outgrowth by the immune system without being eliminated. In most cases, escape from this state requires loss of antigen presentation and consequent loss of immunogenicity. To characterize this pre-outgrowth phase of cancer-immune interaction and test the efficacy of interventions during dormancy, we assembled a genetically-engineered mouse model (GEMM) of inducible, Kras-driven carcinogenesis coupled to a fluorescent reporter. Early characterization of this model revealed a confounding artificial tolerance mechanism that renders inducible alleles automatically non-immunogenic in autochthonous settings. To circumvent this tolerance, we transplant dissociated epidermal keratinocytes from the GEMM onto recipient animals that do not carry the inducible mutant Kras allele. This transplant step also enables the use recipient animals carrying various immune deficiencies. After graft healing, transformation is induced and the grafts are imaged repeatedly over time via intravital confocal microscopy. In immunocompromised recipients, transformation initiates the rapid growth of labeled cells and subsequent tumor formation. In immunocompetent cohorts, tumor formation does not occur. Instead, we observe establishment of a long-term equilibrium phase characterized by cyclic growth and regression of labeled clones originating exclusively from hair follicles, suggesting that the immune privilege of this stem cell niche provides a safe harbor for transformed cells. Surprisingly, the architecture of the normal tissue defines a protected reservoir for the survival of transformed cells, a fate that usually requires cell-intrinsic immune-evasive adaptations. This provides a mechanism by which a single source of anti-tumor activity could produce the observed biphasic nature of cancer-immune interaction, including both elimination of some clones and immune-mediated dormancy of others. Most importantly, we have identified a novel pre-malignant, immune-controlled state that may represent a vulnerable target population for rational cancer prevention strategies. Citation Format: Brad Kubick, Dennis Roop. Epidermal neoplasms bypass immune evasion requirements via niche occupancy. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR14.

Abstract 4226: The rodent eye as a non-invasive window for understanding cancer nanotherapeutics

Abstract We have successfully used the mouse eye as a non-surgical window for highly efficient, optical investigation of xenograft models, using a state-of-the-art ocular imaging facility, the UC Davis “EyePod”. The EyePod employs single-cell resolution intravital confocal microscopy and optical coherence tomography, performed completely non-invasively through the natural optics of the eye (1). This technology enables repeatable in vivo imaging over days, weeks and even months, quantitative tracking of tumor development and delivery of theranostic nanoparticles, and the measurement of tumor and tumor microenvironment responses. Moreover, the retina is a highly “approachable part of the brain”, so that non-invasive study of ocular tumors provides a platform for examining such critical issues as drug delivery across the blood retinal barrier (BRB) and blood brain barrier (BBB). Clinical metastasis of solid tumor to the uvea is not uncommon. Therefore, uvea/subretinal xenograft implant, even though not orthotopic, is clinically relevant. To visualize and photo-manipulate nanoparticle delivery to intraocular xenograft, we have used our recently reported nanoporphyrin (2) as the model system. This novel multifunctional porphyrin-based micellar nanoplatform allows (i) efficient encapsulation of hydrophobic chemotherapeutic drugs or fluorescent dyes, (ii) near-infra red fluorescent (NIRF) detection of the tumor via the intrinsic fluorescence of porphyrins, (iii) photodynamic therapy (PDT) and photothermal therapy (PTT) via efficient free radical and heat generation at the tumor site, respectively. Thorough understanding of how this nanocarrier distributes within the tumor microenvironment, and how it responds to controlled optical stimulation will enable us to maximize its therapeutic potential as a nano-theranostic agent. Preliminary FRET study with doxorubicin-loaded nanoporphyrin has allowed us to visualize in vivo and in real time the release of doxorubicin at the tumor site upon illumination with 680nm laser. In addition, the EyePod has enabled us to follow the tumor response over a period of 35 days. Work is currently underway to (i) develop patient-derived xenograft (PDX) model in the eye of NSG mice, and (ii) use EyePod to study the effect of tumor targeting and brain endothelial cell targeting peptides, recently developed in our laboratory, for nanodelivery into the tumor cells and across the BRB. Citation Format: Xinlei Wang, Wenwu Xiao, Yuanpei Li, Pengfei Zhang, Mayank Goswami, Robert J. Zawadzki, Edward N. Pugh, Kit S. Lam. The rodent eye as a non-invasive window for understanding cancer nanotherapeutics. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4226.

The Borrelia burgdorferi CheY3 response regulator is essential for chemotaxis and completion of its natural infection cycle.

Borrelia burgdorferi possesses a sophisticated and complex chemotaxis system, but how the organism utilizes this system in its natural enzootic life cycle is poorly understood. Of the three CheY chemotaxis response regulators in B. burgdorferi, we found that only deletion of cheY3 resulted in an altered motility and significantly reduced chemotaxis phenotype. Although ΔcheY3 maintained normal densities in unfed ticks, their numbers were significantly reduced in fed ticks compared with the parental or cheY3-complemented spirochetes. Importantly, mice fed upon by the ΔcheY3-infected ticks did not develop a persistent infection. Intravital confocal microscopy analyses discovered that the ΔcheY3 spirochetes were motile within skin, but appeared unable to reverse direction and perform the characteristic backward-forward motility displayed by the parental strain. Subsequently, the ΔcheY3 became 'trapped' in the skin matrix within days of inoculation, were cleared from the skin needle-inoculation site within 96 h post-injection and did not disseminate to distant tissues. Interestingly, although ΔcheY3 cells were cleared within 96 h post-injection, this attenuated infection elicited significant levels of B. burgdorferi-specific IgM and IgG. Taken together, these data demonstrate that cheY3-mediated chemotaxis is crucial for motility, dissemination and viability of the spirochete both within and between mice and ticks.

Identification and treatment of the Staphylococcus aureus reservoir in vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC's antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

Neutrophil extracellular traps (NETs) promote disseminated intravascular coagulation in sepsis.

Abstract Neutrophil extracellular traps (NETs; webs of DNA coated in anti-microbial proteins) are released into the vasculature during sepsis and contribute to organ damage. Various components of NETs have been shown to modulate the coagulation cascade in vitro. We hypothesized that NETs released into the vasculature during sepsis activate intravascular coagulation, leading to microvascular hypoperfusion and end-organ damage. Multi-color confocal intravital microscopy was used in mouse models of sepsis (endotoxemia and E. coli peritonitis) to visualize and quantify neutrophil and platelet trafficking, NETs production, real-time perfusion analysis, and intravascular thrombin activity (using novel in vivo zymography). In vivo imaging of the liver microcirculation in septic mice revealed spatial co-localization between NETs and intravascular coagulation (thrombin activity, fibrin deposition, and platelet aggregation). Inhibition of NETs using i.v. DNase infusion (to digest NETs), or PAD4-deficient mice (that have impaired NETs production) resulted in significantly lower quantities of intravascular thrombin activity, and improved microvascular perfusion. In a model of Gram-negative sepsis (E. coli peritonitis), PAD4-deficient mice had reduced markers of disseminated intravascular coagulation (intravascular thrombin, PAI-1), and end-organ damage (serum lactate, ALT) compared to wild-type controls. Together, these data demonstrate that NETs activate coagulation in vivo, and promote disseminated intravascular coagulation in sepsis. Inhibition of NETs in septic animals reduces intravascular coagulation, improves microvascular perfusion, and attenuates end-organ damage.

Modern demodicosis diagnostics methods

An overview of the modern demodicosis diagnostics methods and in-house demodicosis diagnosis with intravital laser scanning confocal microscopy effectiveness studies. The study included 60 patients with acne and rosacea, complicated demodicosis, 60 patients with acne and rosacea and no demodicosis, and 30 healthy volunteers. All the patients underwent skin scraping, eyebrows and/or eyelashes hair removal and confocal laser-scanning microscopy. the advantages of confocal laser-scanning microscopy in vivo in comparison with conventional methods of research are shown.

Vascular bursts enhance permeability of tumour blood vessels and improve nanoparticle delivery.

Enhanced permeability in tumours is thought to result from malformed vascular walls with leaky cell-to-cell junctions. This assertion is backed by studies using electron microscopy and polymer casts that show incomplete pericyte coverage of tumour vessels and the presence of intercellular gaps. However, this gives the impression that tumour permeability is static amid a chaotic tumour environment. Using intravital confocal laser scanning microscopy we show that the permeability of tumour blood vessels includes a dynamic phenomenon characterized by vascular bursts followed by brief vigorous outward flow of fluid (named 'eruptions') into the tumour interstitial space. We propose that 'dynamic vents' form transient openings and closings at these leaky blood vessels. These stochastic eruptions may explain the enhanced extravasation of nanoparticles from the tumour blood vessels, and offer insights into the underlying distribution patterns of an administered drug.

Visualizing the Tumor Microenvironment of Liver Metastasis by Spinning Disk Confocal Microscopy.

Intravital microscopy has evolved into an invaluable technique to study the complexity of tumors by visualizing individual cells in live organisms. Here, we describe a method for employing intravital spinning disk confocal microscopy to picture high-resolution tumor-stroma interactions in real time. We depict in detail the surgical procedures to image various tumor microenvironments and different cellular components in the liver.

The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis invivo. Wedemonstrated that the Bruton's tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation.

A New Model for Studying the Dissemination of Myeloma Cells throughout the Bone Marrow Using Embryonic Zebrafish

Multiple myeloma develops from a pre-malignant clonal proliferation of plasma cells. The dissemination of myeloma cells throughout the bone marrow (BM) is an important early step in myeloma pathogenesis. Studies of myeloma cell homing in mouse models are not quantitative and cannot be used for functional genomics or drug screening. To overcome these limitations, we have developed a novel in vivo model to assess multiple myeloma (MM) cell homing, that takes advantage of the optical clarity of zebrafish (Danio rerio) embryos.We performed intra-cardiac (i.c.) injection of GFP+MM.1S cells into zebrafish embryos 48 hpf, and assessed the ability of the injected cells to enter the peripheral circulation and then traced their homing to the trunk region of zebrafish embryos, an area referred as the caudal hematopoietic tissue (CHT), by using intra-vital confocal microscopy. We next injected primary CD138+ cells derived from MM patient BM that had been stained with either DiO or DiD and demonstrated that they also homed to CHT. As a control, we injected DiO-labeled CD41-GFPlow zebrafish hematopoietic stem cells (HSCs) that are known to home to the CHT hematopoietic niche; and observed that zebrafish-derived CD41-HSCs homed to the same area as MM cells.We next analyzed changes in the transcriptome of those MM cells that homed to the CHT-niche. We dissected the zebrafish embryos to separate the CHT from other tissue containing non-adherent MM cells and performed whole human exome enrichment prior to sequencing of total RNA. We had an alignment rate of 10-15%, with a high intragenic rate an exonic rate (> 95%) and a low mismatch rate (~0.5%). RNAseq revealed that the MM cells that homed to the CHT were enriched in transcripts important for cytokine/chemokine mediated signaling, the IL-6 signalling pathway, cell-cell adhesion and angiogenesis (FDR<0.25; P<0.05). Overall, these findings indicate that the changes observed in MM cells that have homed to the CHT mirror those that are seen in MM cells that are resident in the human BM. In order to investigate the functional relevance of the zebrafish model, we established CXCR4-, VLA-4- and FAK-silenced MM cells and compared their ability to home to CHT to that of control scrambled shRNA-transfected cells. DiO-labeled-CXCR4-silenced and DiO-labeled-scrambled-probe control MM cells were mixed in equal numbers and subsequently injected into recipient zebrafish. We found a significant reduction in the number of CXCR4-silenced MM cells homing to the CHT, compared to the control cells (P<0.00). We then examined VLA-4- and FAK-knock-down MM cells and observed that the homing of MM cells to CHT was impaired when either VLA-4 or FAK were silenced (P<0.001; Fig. 3C-D). Having demonstrated the role of CXCF4, FAK and VLA4 in MM cell homing to the CHT niche, we next performed qRT-PCR for those transcripts and confirmed that MM cells harvested from the CHT areas expressed higher levels of CXCR4, FAK and VLA4, compared to MM cells harvested from non-CHT areas. (P<0.05). To ascertain whether homing to zebrafish embryo CHT is occurs in other hematologic malignancies that are known to home to the human and murine BM, we used a cultured cell lined derived from a patient with Waldenstrom's Macroglobulinemia (WM). We injected either CXCR4-overexpressing or CXCR4-silenced WM cells and found that increased CXCR4 expression in WM cells led to enhanced CHT-homing of WM cells (P<0.001), while the homing of CXCR4-silenced WM cells to the CHT was reduced compared to scrambled control (P<0.001).These findings demonstrate that zebrafish can be used to study the homing of human myeloma cells to a hematopoietic niche. The rapidity of homing to CHT, which occurs within seconds of cell injection, suggests that a fraction of the CD138+ harvested from patient bone marrow already express those RNA transcripts and proteins needed for stable adhesion and residence in CHT. This hypothesis is confirmed by the RNA seq and qRT-PCR studies which directly demonstrate increased expression of relevant transcripts in adherent cells. This zebrafish model may provide new insights into the pathogenesis of MM and may be useful as a means to screen for agents which can disrupt homing and dissemination of MM cells. DisclosuresNo relevant conflicts of interest to declare.

Systemically Injectable Enzyme‐Loaded Polyion Complex Vesicles as In Vivo Nanoreactors Functioning in Tumors

AbstractThe design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid‐ and polymeric‐vesicle‐based nanoreactors have some practical limitations. We have succeeded in preparing enzyme‐loaded polyion complex vesicles (PICsomes) through a facile protein‐loading method. The preservation of enzyme activity was confirmed even after cross‐linking of the PICsomes. The cross‐linked β‐galactosidase‐loaded PICsomes (β‐gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER‐βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β‐gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme‐loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.

Systemically Injectable Enzyme-Loaded Polyion Complex Vesicles as In Vivo Nanoreactors Functioning in Tumors.

The design and construction of nanoreactors are important for biomedical applications of enzymes, but lipid- and polymeric-vesicle-based nanoreactors have some practical limitations. We have succeeded in preparing enzyme-loaded polyion complex vesicles (PICsomes) through a facile protein-loading method. The preservation of enzyme activity was confirmed even after cross-linking of the PICsomes. The cross-linked β-galactosidase-loaded PICsomes (β-gal@PICsomes) selectively accumulated in the tumor tissue of mice. Moreover, a model prodrug, HMDER-βGal, was successfully converted into a highly fluorescent product, HMDER, at the tumor site, even 4 days after administration of the β-gal@PICsomes. Intravital confocal microscopy showed continuous production of HMDER and its distribution throughout the tumor tissues. Thus, enzyme-loaded PICsomes are useful for prodrug activation at the tumor site and could be a versatile platform for enzyme delivery in enzyme prodrug therapy.

Platelet aggregation but not activation and degranulation during the acute post-ischemic reperfusion phase in livers with no underlying disease

Platelets and P-selectin (CD62P) play an unequivocal role in the pathology of hepatic ischemia/reperfusion (I/R) injury. Inhibition or knock-out of P-selectin or immunodepletion of platelets results in amelioration of post-ischemic inflammation, reduced hepatocellular damage, and improved survival. However, P-selectin expression on platelets and endothelial cells, which concurs with platelet activation, has never been clearly demonstrated in I/R-subjected livers. To determine whether platelets become activated and degranulate in the acute phase of liver I/R and whether the platelets interact with neutrophils. Hepatic I/R was induced in male C57BL/6J mice (N = 12) using 37.5-min ischemia time. Platelets, endothelial cells, and neutrophils were fluorescently labeled by systemic administration of non-blocking antibodies. Cell kinetics were monitored by intravital spinning disk confocal microscopy during 90 min of reperfusion. Image analysis and quantification was performed with dedicated software. Platelets adhered to sinusoids more extensively in post-ischemic livers compared to livers not subjected to I/R and formed aggregates, which occurred directly after ischemia. Platelets and endothelial cells did not express P-selectin in post-ischemic livers. There was no interaction between platelets and neutrophils. Platelets aggregate but do not become activated and do not degranulate in post-ischemic livers. There is no platelet-neutrophil interplay during the early reperfusion phase in a moderate model of hepatic I/R injury. The mechanisms underlying the biological effects of platelets and P-selectin in this setting warrant further investigation. I/R in surgical liver patients may compromise outcome due to post-ischemic oxidative stress and sterile inflammation. Both processes are mediated in part by platelets. Understanding platelet function during I/R is key to developing effective interventions for I/R injury and improving clinical outcomes.

Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 333 Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire Mariko Kurotsu, Emiyu Ogawa, Tsunenori Arai Graduate School of Science and Technology, Keio University, Japan Westudied theelectrical conduction (EC)block recoverybyphotosensitization reaction (PR) under extracellular talaporfin sodium existence in a novel cardiomyocyte EC wire in vitro. Tachyarrhythmia can be cured by permanent block of abnormal EC. The EC block recovery sometimes occurs in the case of weak damage on myocardium. The pattern cultivation cover glass, which had 60 m width cultivation areas, was used to form the wires. The PR was operated to the wires for 600 s varying 3–120mW/cm2 in 663nm laser irradianceswith 20 g/ml talaporfin sodium. The cardiomyocyte ECwas evaluated by the cross correlation function of a fluorescence brightness of intracellular Ca2+ probe in a wire. In 30 and 60mW/cm2, the EC of 15min after the PR was blocked significantly faster than the reported cardiomyocyte necrosis occurrence timing of the same PR conditions. Despite the EC block probabilities of 15min and 2h after the PR were almost 90% in 60mW/cm2, those in 30mW/cm2 were approximately 90 and 60%, respectively. We think this temporary EC block might be caused by gap junction closure induced by intracellular Ca2+ increase. http://dx.doi.org/10.1016/j.pdpdt.2015.07.036 Light fractionation improves the response to BF-200 ALA-PDT in hairless mouse skin Henriette S. de Bruijn1, Sander Brooks2, Angelique van der Ploeg-van den Heuvel1, Ellen R.M. de Haas2, Dominic J. Robinson1 1 The Centre for Optical Diagnostics and Therapy, Department of Otolaryngology and Head & Neck Surgery, The Netherlands 2 Department of Dermatology, Erasmus MC, Rotterdam, The Netherlands Fractionated illumination significantly enhances the effectiveness of 5-aminolevulinic-acid based photodynamic therapy (ALAPDT) but not of Methyl-AminoLevulinate (MAL). BF-200 ALA is a recently approved novel formulation of 10% ALA in a lecithin-based nanoemulsion. Here we compare the fluorescence kinetics, distribution and PDT induced response in hairless mice using BF-200 ALA, MAL and ALA. Comparable fluorescence kinetics was determined forMAL, ALAandBF-200ALA. Cryo-sectionswere stained for CD31 and co-localization with PpIX fluorescence was significantly stronger after ALA and BF-200 ALA compared to MAL (p<0.01). Intra-vital confocalmicroscopy showed no difference betweenALA and BF-200 ALA at depth in skin. Vascular responses were not different as determined in the skin-fold chamber immediately after PDT or at day 1 in cryosections. Visual skin damage scored up to 14 days post PDT showed an significantly increased response to light fractionation for bothALAandBF-200ALA. BF-200ALAbehaves like ALA in normal mouse skin. While light fractionated ALA-PDT was successfully translated to a treatment modality for human lesions our results suggest that ALA can be replaced by BF-200 ALA, marketed as Ameluz® in Europe, for the treatment of superficial BasalCell-Carcinoma and other skin lesions using light fractionation. http://dx.doi.org/10.1016/j.pdpdt.2015.07.037 Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 333 Conduction block recovery by photosensitization reaction under extracellular talaporfin sodium existence in a cardiomyocyte electrical conduction wire Mariko Kurotsu, Emiyu Ogawa, Tsunenori Arai Graduate School of Science and Technology, Keio University, Japan Westudied theelectrical conduction (EC)block recoverybyphotosensitization reaction (PR) under extracellular talaporfin sodium existence in a novel cardiomyocyte EC wire in vitro. Tachyarrhythmia can be cured by permanent block of abnormal EC. The EC block recovery sometimes occurs in the case of weak damage on myocardium. The pattern cultivation cover glass, which had 60 m width cultivation areas, was used to form the wires. The PR was operated to the wires for 600 s varying 3–120mW/cm2 in 663nm laser irradianceswith 20 g/ml talaporfin sodium. The cardiomyocyte ECwas evaluated by the cross correlation function of a fluorescence brightness of intracellular Ca2+ probe in a wire. In 30 and 60mW/cm2, the EC of 15min after the PR was blocked significantly faster than the reported cardiomyocyte necrosis occurrence timing of the same PR conditions. Despite the EC block probabilities of 15min and 2h after the PR were almost 90% in 60mW/cm2, those in 30mW/cm2 were approximately 90 and 60%, respectively. We think this temporary EC block might be caused by gap junction closure induced by intracellular Ca2+ increase. http://dx.doi.org/10.1016/j.pdpdt.2015.07.036 Light fractionation improves the response to BF-200 ALA-PDT in hairless mouse skin Henriette S. de Bruijn1, Sander Brooks2, Angelique van der Ploeg-van den Heuvel1, Ellen R.M. de Haas2, Dominic J. Robinson1 1 The Centre for Optical Diagnostics and Therapy, Department of Otolaryngology and Head & Neck Surgery, The Netherlands 2 Department of Dermatology, Erasmus MC, Rotterdam, The Netherlands Fractionated illumination significantly enhances the effectiveness of 5-aminolevulinic-acid based photodynamic therapy (ALAPDT) but not of Methyl-AminoLevulinate (MAL). BF-200 ALA is a recently approved novel formulation of 10% ALA in a lecithin-based nanoemulsion. Here we compare the fluorescence kinetics, distribution and PDT induced response in hairless mice using BF-200 ALA, MAL and ALA. Comparable fluorescence kinetics was determined forMAL, ALAandBF-200ALA. Cryo-sectionswere stained for CD31 and co-localization with PpIX fluorescence was significantly stronger after ALA and BF-200 ALA compared to MAL (p<0.01). Intra-vital confocalmicroscopy showed no difference betweenALA and BF-200 ALA at depth in skin. Vascular responses were not different as determined in the skin-fold chamber immediately after PDT or at day 1 in cryosections. Visual skin damage scored up to 14 days post PDT showed an significantly increased response to light fractionation for bothALAandBF-200ALA. BF-200ALAbehaves like ALA in normal mouse skin. While light fractionated ALA-PDT was successfully translated to a treatment modality for human lesions our results suggest that ALA can be replaced by BF-200 ALA, marketed as Ameluz® in Europe, for the treatment of superficial BasalCell-Carcinoma and other skin lesions using light fractionation. http://dx.doi.org/10.1016/j.pdpdt.2015.07.037 Parameter study of myocardial cell damage with photosensitization reaction under extracellular talaporfin sodium existence

Abstract 4180: TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is projected to become the 2nd leading cause of cancer deaths by 2030. PDACs are associated with a high frequency of KRAS mutations (95%) and overexpression of many pro-angiogenic cytokines and growth factors. Using a genetically engineered mouse model (GEMM) that we established in which oncogenic Kras is combined with loss of RB (KRC), we have determined that PDACs arising in these mice (mPDACs) harbor many endothelial cells (ECs) and sinusoidal-like vessels that have blood flow, as determined by intravital confocal microscopy. Array analysis of pancreatic cancer cells (PCCs) derived from KRC tumors revealed gene expression profiles that correlate with active TGF-β signaling pathways, as determined by gene set enrichment analysis (GSEA), and include increased expression of multiple pro-angiogenic genes. In silico analysis indicated that many of these pro-angiogenic genes were TGF-β targets, and inhibition of the type I TGF-β receptor (TβRI) with SB505124 confirmed that TGF-βs drive pro-angiogenic gene expression in KRC PCCs. Moreover, TGF-β increased the levels of pro-angiogenic cytokines in conditioned media (CM) prepared from KRC PCCs, which when added to ECs, activated canonical TGF-β signaling pathways and stimulated EC proliferation and migration. Although SB505124 blocked TGF-β pathway activation in ECs, it failed to suppress CM-enhanced EC proliferation and migration. By contrast, SB505124 attenuated tumor angiogenesis, growth and metastasis in a syngeneic orthotopic mouse model using KRC PCCs. Taken together, these results suggest that TGF-β promotes angiogenesis in an indirect manner, by up-regulating pro-angiogenic factors in PCCs that act on ECs in a paracrine manner. Therefore, targeting TGF-β in PDAC could be a beneficial anti-angiogenic strategy. Citation Format: Jesse Gore, Kelly E. Craven, Julie L. Wilson, Murray Korc. TGF-beta promotes angiogenesis in an RB-deficient, Kras-driven mouse model of pancreatic cancer. [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 4180. doi:10.1158/1538-7445.AM2015-4180

Smart imaging of acute lung injury: exploration of myeloperoxidase activity using in vivo endoscopic confocal fluorescence microscopy.

The pathophysiology of acute lung injury (ALI) is well characterized, but its real-time assessment at bedside remains a challenge. When patients do not improve after 1 wk despite supportive therapies, physicians have to consider open lung biopsy (OLB) to identify the process(es) at play. Sustained inflammation and inadequate repair are often observed in this context. OLB is neither easy to perform in a critical setting nor exempt from complications. Herein, we explore intravital endoscopic confocal fluorescence microscopy (ECFM) of the lung in vivo combined with the use of fluorescent smart probe(s) activated by myeloperoxidase (MPO). MPO is a granular enzyme expressed by polymorphonuclear neutrophils (PMNs) and alveolar macrophages (AMs), catalyzing the synthesis of hypoclorous acid, a by-product of hydrogen peroxide. Activation of these probes was first validated in vitro in relevant cells (i.e., AMs and PMNs) and on MPO-non-expressing cells (as negative controls) and then tested in vivo using three rat models of ALI and real-time intravital imaging with ECFM. Semiquantitative image analyses revealed that in vivo probe-related cellular/background fluorescence was associated with corresponding enhanced lung enzymatic activity and was partly prevented by specific MPO inhibition. Additional ex vivo phenotyping was performed, confirming that fluorescent cells were neutrophil elastase(+) (PMNs) or CD68(+) (AMs). This work is a first step toward "virtual biopsy" of ALI without OLB.

Differential effects of α4β7 and GPR15 on homing of effector and regulatory T cells from patients with UC to the inflamed gut in vivo

ObjectiveGut homing of lymphocytes via adhesion molecules has recently emerged as new target for therapy in IBDs. We aimed to analyse the in vivo homing of effector (Teff) and regulatory...

Analyzing Structure and Function of Vascularization in Engineered Bone Tissue by Video-Rate Intravital Microscopy and 3D Image Processing.

Vascularization is a key challenge in tissue engineering. Three-dimensional structure and microcirculation are two fundamental parameters for evaluating vascularization. Microscopic techniques with cellular level resolution, fast continuous observation, and robust 3D postimage processing are essential for evaluation, but have not been applied previously because of technical difficulties. In this study, we report novel video-rate confocal microscopy and 3D postimage processing techniques to accomplish this goal. In an immune-deficient mouse model, vascularized bone tissue was successfully engineered using human bone marrow mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells (HUVECs) in a poly (D,L-lactide-co-glycolide) (PLGA) scaffold. Video-rate (30 FPS) intravital confocal microscopy was applied in vitro and in vivo to visualize the vascular structure in the engineered bone and the microcirculation of the blood cells. Postimage processing was applied to perform 3D image reconstruction, by analyzing microvascular networks and calculating blood cell viscosity. The 3D volume reconstructed images show that the hMSCs served as pericytes stabilizing the microvascular network formed by HUVECs. Using orthogonal imaging reconstruction and transparency adjustment, both the vessel structure and blood cells within the vessel lumen were visualized. Network length, network intersections, and intersection densities were successfully computed using our custom-developed software. Viscosity analysis of the blood cells provided functional evaluation of the microcirculation. These results show that by 8 weeks, the blood vessels in peripheral areas function quite similarly to the host vessels. However, the viscosity drops about fourfold where it is only 0.8 mm away from the host. In summary, we developed novel techniques combining intravital microscopy and 3D image processing to analyze the vascularization in engineered bone. These techniques have broad applicability for evaluating vascularization in other engineered tissues as well.