Tatiana B. Krasieva

Polarity of T Cell Shape, Motility, and Sensitivity to Antigen

T cell activation requires contact with APCs. We used optical techniques to demonstrate T cell polarity on the basis of shape, motility, and localized sensitivity to antigen. An intracellular Ca2+ clamp showed that T cell shape and motility are extremely sensitive to changes in [Ca2+]i (Kd = 200 nM), with immobilization and rounding occurring via a calcineurin-independent pathway. Ca2+ dependent immobilization prolonged T cell contact with the antigen-presenting B cell; buffering the [Ca2+]i signal prevented the formation of stable cell pairs. Optical tweezers revealed spatial T cell sensitivity to antigen by controlling placement on the T cell surface of either B cells or alpha-CD3 MAb-coated beads. T cells were 4-fold more sensitive to contact made at the leading edge of the T cell compared with the tail. We conclude that motile T cells are polarized antigen sensors that respond physically to [Ca2+]i signals to stabilize their interaction with APCs.

Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells

Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.

Genome-wide siRNA-based functional genomics of pigmentation identifies novel genes and pathways that impact melanogenesis in human cells.

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinsons disease), auditory disorders (Waardenburgs syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.

UNC-51/ATG1 kinase regulates axonal transport by mediating motor–cargo assembly

Axonal transport mediated by microtubule-dependent motors is vital for neuronal function and viability. Selective sets of cargoes, including macromolecules and organelles, are transported long range along axons to specific destinations. Despite intensive studies focusing on the motor machinery, the regulatory mechanisms that control motor-cargo assembly are not well understood. Here we show that UNC-51/ATG1 kinase regulates the interaction between synaptic vesicles and motor complexes during transport in Drosophila. UNC-51 binds UNC-76, a kinesin heavy chain (KHC) adaptor protein. Loss of unc-51 or unc-76 leads to severe axonal transport defects in which synaptic vesicles are segregated from the motor complexes and accumulate along axons. Genetic studies show that unc-51 and unc-76 functionally interact in vivo to regulate axonal transport. UNC-51 phosphorylates UNC-76 on Ser(143), and the phosphorylated UNC-76 binds Synaptotagmin-1, a synaptic vesicle protein, suggesting that motor-cargo interactions are regulated in a phosphorylation-dependent manner. In addition, defective axonal transport in unc-76 mutants is rescued by a phospho-mimetic UNC-76, but not a phospho-defective UNC-76, demonstrating the essential role of UNC-76 Ser(143) phosphorylation in axonal transport. Thus, our data provide insight into axonal transport regulation that depends on the phosphorylation of adaptor proteins.

Photothermal treatment of glioma; an in vitro study of macrophage-mediated delivery of gold nanoshells

One of the major factors that limits the treatment effectiveness for gliomas is the presence of the blood–brain barrier (BBB) which protects infiltrating glioma cells from the effects of anti-cancer agents. Circulating monocytes/macrophages (Ma) have a natural ability to traverse the intact and compromised BBB and loaded with anti cancer agents could be used as vectors to target tumors and surrounding tumor infiltrated tissue. Nanoshells (NS) are composed of a dielectric core (silica) coated with an ultrathin gold layer which converts absorbed near-infrared light (NIR) to heat with an extremely high efficacy and stability. We have investigated the effects of exposure to laser NIR on multicell human glioma spheroids infiltrated with empty (containing no nanoshells) or nanoshell loaded macrophages. Our results demonstrated that; (1) macrophages could efficiently take up bare or coated (PEGylated) gold NS: (2) NS loaded macrophages infiltrated into glioma spheroids to the same or, in some cases, to a greater degree than empty Ma; (3) NIR laser irradiation of spheroids incorporating NS loaded macrophages resulted in complete growth inhibition in an irradiance dependent manner, and (4) spheroids infiltrated with empty macrophages had growth curves identical to untreated control cultures. The results of this study provide proof of concept for the use of macrophages as a delivery vector of NS into gliomas for photothermal ablation and open the possibility of developing such regimens for patient treatment.

Macrophages as Cell-Based Delivery Systems for Nanoshells in Photothermal Therapy

Site-specific delivery of nanoparticles poses a significant challenge, especially in the brain where the blood–brain barrier prevents the entry of most therapeutic compounds including nanoparticle-based anti-cancer agents. In this context, the use of macrophages as vectors for the delivery of gold–silica nanoshells to infiltrating gliomas will be reviewed in this article. Gold–silica nanoshells are readily phagocytosed by macrophages without any apparent toxic effects, and the results of in vitro studies have demonstrated the migratory potential of nanoshell-loaded macrophages in human glioma spheroids. Of particular interest is the observation that, after near-infrared exposure of spheroids containing nanoshell-loaded macrophages, sufficient heat was generated to suppress spheroid growth. Collectively, these findings demonstrate the potential of macrophages as nanoshell delivery vectors for photothermal therapy of gliomas, and they certainly provide the basis for future animal studies.

Selective photosensitizer distribution in vulvar condyloma acuminatum after topical application of 5-aminolevulinic acid

OBJECTIVE Our purpose was to determine the feasibility of selective photosensitization of vulvar condylomas by use of tropical application of 5-aminolevulinic acid. STUDY DESIGN In vivo fluorescence was assessed and biopsy specimens of condylomas were taken for fluorescence microscopy in 24 patients at different times after application of 2.5% 5-aminolevulinic acid ointment or 20% 5-aminolevulinic acid cream. RESULTS Both in vivo fluorescence imaging and fluorescence microscopy showed selective fluorescence of condylomas of the labia minora and vestibule only within short time intervals, because fluorescence of poorly keratinized normal epithelium was induced by both 5-aminolevulinic acid formulations. In non-hair-bearing skin, lesional fluorescence remained highly selective. Fluorescence microscopy showed that 90 minutes after drug application peak selectivity in epithelial lesional fluorescence was significantly higher with 2.5% 5-aminolevulinic acid ointment (4.5 +/- 0.9) than it was with 20% cream (2.1 +/- 0.2). CONCLUSION Selective fluorescence of vulvar condyloma acuminatum can be induced by nonselective topical 5-aminolevulinic acid application. Studies evaluating selective photodynamic destruction of condylomas are justified.

Noninvasive imaging of angiotensin receptors after myocardial infarction.

OBJECTIVES The purpose of this study was to evaluate the feasibility of noninvasive imaging of angiotensin II (AT) receptor upregulation in a mouse model of post-myocardial infarction (MI) heart failure (HF). BACKGROUND Circulating AT levels do not reflect the status of upregulation of renin-angiotensin axis in the myocardium, which plays a central role in ventricular remodeling and evolution of HF after MI. Appropriately labeled AT or AT receptor blocking agents should be able to specifically target AT receptors by molecular imaging techniques. METHODS AT receptor imaging was performed in 29 mice at various time points after permanent coronary artery ligation or in controls using a fluoresceinated angiotensin peptide analog (APA) and radiolabeled losartan. The APA was used in 19 animals for intravital fluorescence microscopy on a beating mouse heart. Tc-99m losartan was used for in vivo radionuclide imaging and quantitative assessment of AT receptor expression in 10 mice. After imaging, hearts were harvested for pathological characterization using confocal and 2-photon microscopy. RESULTS No or little APA uptake was observed in control animals or within infarct regions on days 0 and 1. Distinct uptake occurred in the infarct area at 1 to 12 weeks after MI; the uptake was at maximum at 3 weeks and reduced markedly at 12 weeks after MI. Ultrasonographic examination demonstrated left ventricular remodeling, and pathologic characterization revealed localization of the APA tracer with collagen-producing myofibroblasts. Tc-99m losartan uptake in the infarct region (0.524 +/- 0.212% injected dose/g) increased 2.4-fold as compared to uptake in the control animals (0.215 +/- 0.129%; p < 0.05). CONCLUSIONS The present study demonstrates the feasibility of in vivo molecular imaging of AT receptors in the remodeling myocardium. Noninvasive imaging studies aimed at AT receptor expression could play a role in identification of subjects likely to develop heart failure. In addition, such a strategy could allow for optimization of anti-angiotensin therapy in patients after MI.

Three-Dimensional Spheroid Culture of Human Gingiva-Derived Mesenchymal Stem Cells Enhances Mitigation of Chemotherapy-Induced Oral Mucositis

Mesenchymal stem cells (MSCs) are capable of regenerative and immunomodulatory functions in cell-based therapies in a variety of human diseases and injuries; however, their therapeutic efficacy and potential side effects remain major obstacles in clinical applications. We report here a 3D spheroid culture approach to optimize stem cell properties and therapeutic effects of human gingiva-derived mesenchymal stem cells (GMSCs) in mitigation of experimental oral mucositis. Under growth condition of ultra-low attachment, GMSCs spontaneously aggregated into 3D spheroids and exhibited distinct early stem cell phenotype characterized by elevated expression Stro-1 and CXC chemokine receptor 4 (CXCR-4) as well as OCT-4 and Nanog, 2 important transcriptional factors relevant to stem cell properties, and decreased expression of MSC-associated markers, including CD29, CD90, and CD105. Functionally, spheroid GMSCs are capable of enhanced multipotency and augmented secretion of several chemokines and cytokines relevant to cell migration, survival, and angiogenesis. More importantly, spheroid GMSCs expressed increased levels of reactive oxygen species, hypoxia-inducible factor (HIF)-1 and -2α, and manganese superoxide dismutase, which correlated with improved resistance to oxidative stress-induced apoptosis. Using an in vivo murine model of chemotherapy-induced oral mucositis, we demonstrated that spheroid-derived GMSCs possessed better therapeutic efficacy than their adherent cells in reversing body weight loss and promoting the regeneration of disrupted epithelial lining of the mucositic tongues. These findings suggest that 3D spheroid culture allows early stemness preservation and potentially precondition GMSCs for enhanced mitigation of oral mucositis.

Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media

We present a comparative study of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging in turbid media at 800- and 1300-nm excitation. The depth-dependent decay of TPEF and SHG signals in turbid tissue phantoms is used to estimate the impact of light scattering on excitation intensity at each wavelength. A 50 to 80% increase in scattering length is observed using 1300-nm excitation, while peak TPEF emission intensity is obtained 10 to 20 microm beneath the surface for both sources. The increased penetration depth at 1300 nm is confirmed by TPEF and SHG microscopy of tissue phantoms composed of gelatin/microspheres and 3-D organotypic collagen-fibroblast cultures, respectively. Our results establish the feasibility of 1.3-microm excitation in nonlinear optical microscopy.