Elisa Carrasco

1.3 μm emitting SrF2:Nd3+ nanoparticles for high contrast in vivo imaging in the second biological window

Novel approaches for high contrast, deep tissue, in vivo fluorescence biomedical imaging are based on infrared-emitting nanoparticles working in the so-called second biological window (1,000–1,400 nm). This allows for the acquisition of high resolution, deep tissue images due to the partial transparency of tissues in this particular spectral range. In addition, the optical excitation with low energy (infrared) photons also leads to a drastic reduction in the contribution of autofluorescence to the in vivo image. Nevertheless, as is demonstrated here, working solely in this biological window does not ensure a complete removal of autofluorescence as the specimen’s diet shows a remarkable infrared fluorescence that extends up to 1,100 nm. In this work, we show how the 1,340 nm emission band of Nd3+ ions embedded in SrF2 nanoparticles can be used to produce autofluorescence free, high contrast in vivo fluorescence images. It is also demonstrated that the complete removal of the food-related infrared autofluorescence is imperative for the development of reliable biodistribution studies.

Protoporphyrin IX-dependent photodynamic production of endogenous ROS stimulates cell proliferation.

Photodynamic therapy using methyl 5-aminolevulinate (MAL) as a precursor of the photosensitizing agent protoporphyrin IX is widely used in clinical practice for the treatment of different pathologies, including cancer. In this therapeutic modality, MAL treatment promotes the forced accumulation of the endogenous photoactive compound protoporphyrin IX in target malignant cells. Subsequent irradiation of treated tissues with an appropriate visible light source induces the production of reactive oxygen species (ROS) that, once accumulated above a critical level, promote cell death. Here we demonstrate that a photodynamic treatment with low MAL concentrations can be used to promote a moderate production of endogenous ROS, which efficiently stimulates cell growth in human immortalized keratinocytes (HaCaT). We also show that this proliferative response requires Src kinase activity and is associated to a transient induction of cyclin D1 expression. Taken together, these results demonstrate for the first time that a combination of light and a photoactive compound can be used to modulate cell cycle progression through Src kinase activation and that a moderate intracellular increase of photogenerated ROS efficiently stimulates cell proliferation.

Isolation and characterization of squamous carcinoma cells resistant to photodynamic therapy

Photodynamic therapy (PDT) employing methyl δ‐aminolevulinic acid (Me‐ALA), as a precursor of the photosensitizer protoporphyrin IX (PpIX), is used for the treatment of non melanoma cutaneous cancer (NMCC). However, one of the problems of PDT is the apparition of resistant cell populations. The aim of this study was to isolate and characterize squamous carcinoma cells SCC‐13 resistant to PDT with Me‐ALA. The SCC‐13 parental population was submitted to successive cycles of Me‐ALA‐PDT and 10 resistant populations were finally obtained. In parental and resistant cells there were analyzed the cell morphology (toluidine blue), the intracellular PpIX content (flow cytometry) and its localization (fluorescence microscopy), the capacity of closing wounds (scratch wound assay), the expression of cell‐cell adhesion proteins (E‐cadherin and β‐catenin), cell‐substrate adhesion proteins (β1‐integrin, vinculin and phospho‐FAK), cytoskeleton proteins (α‐tubulin and F‐actin) and the inhibitor of apoptosis protein survivin, in the activated form as phospho‐survivin (indirect immunofluorescence and Western blot). The results obtained indicate that resistant cells showed a more fibroblastic morphology, few differences in intracellular content of the photosensitizer, higher capacity of closing wounds, higher number of stress fibers, more expression of cell‐substrate adhesion proteins and higher expression of phospho‐survivin than parental cells. These distinctive features of the resistant cells can provide decisive information to enhance the efficacy of Me‐ALA applications in clinic dermatology. J. Cell. Biochem. 112: 2266–2278, 2011.

Photoactivation of ROS Production In Situ Transiently Activates Cell Proliferation in Mouse Skin and in the Hair Follicle Stem Cell Niche Promoting Hair Growth and Wound Healing.

The role of reactive oxygen species (ROS) in the regulation of hair follicle (HF) cycle and skin homeostasis is poorly characterized. ROS have been traditionally linked to human disease and aging, but recent findings suggest that they can also have beneficial physiological functions in vivo in mammals. To test this hypothesis, we transiently switched on in situ ROS production in mouse skin. This process activated cell proliferation in the tissue and, interestingly, in the bulge region of the HF, a major reservoir of epidermal stem cells, promoting hair growth, as well as stimulating tissue repair after severe burn injury. We further show that these effects were associated with a transient Src kinase phosphorylation at Tyr416 and with a strong transcriptional activation of the prolactin family 2 subfamily c of growth factors. Our results point to potentially relevant modes of skin homeostasis regulation and demonstrate that a local and transient ROS production can regulate stem cell and tissue function in the whole organism.

Structure–function relationships of Nile blue (EtNBS) derivatives as antimicrobial photosensitizers

The benzophenothiazinium dye EtNBS has previously been tested as a photosensitizer to mediate photodynamic therapy (PDT). It has been employed to kill cancer cells and microbial cells in vitro and to treat tumors and infections in vivo. We synthesized a panel of derivatives substituted at the 1-position of the benzene ring with electron donating or electron withdrawing groups (amino, acetamido and nitro) and tested their production of reactive oxygen species (ROS) and light-mediated killing of two species of Gram-positive and two species of Gram-negative bacteria. All three compounds showed lower fluorescence, lower yield of ROS and less microbial killing than parent EtNBS, while the order of activity (nitro > amino > acetamido) showed that an electron withdrawing substituent was better than electron donating. To test the hypothesis that 1-substitution distorts the planar structure of the conjugated rings we compared two compounds substituted with N-ethylpropylsulfonamido either at the 1-position or at the 4-position. The 4-isomer was significantly more photoactive than the 1-isomer. We also prepared an EtNBS derivative with a guanidinium group attached to the 5-amino group. This compound had high activity against Gram-negative bacteria due to the extra positive charge. Cellular uptake of the compounds by the four bacterial species was also measured and broadly correlated with activity. These results provided three separate pieces of structure-activity relationship data for antimicrobial photosensitizers based on the EtNBS backbone.

Switching on a transient endogenous ROS production in mammalian cells and tissues

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems.

Standard DNA Methylation Analysis in Mouse Epidermis: Bisulfite Sequencing, Methylation-Specific PCR, and 5-Methyl-Cytosine (5mC) Immunological Detection

In mammals, methylation of cytosine C-5 position is a major heritable epigenetic mark on the DNA molecule. Maintenance of proper DNA methylation patterns is a key process during embryo development and in the maintenance of adult tissue homeostasis. The use of experimental procedures based on the chemical modification of cytosine by sodium bisulfite and the development of antibodies recognizing 5mC have essentially contributed to our knowledge on DNA methylation dynamics in normal and disease states. Here we describe standard procedures for bisulfite sequencing, methylation-specific PCR, and 5mC immunodetection using mouse skin and the hair follicle stem cell niche as model tissues.

In-vivo monitoring of infectious diseases in living animals using bioluminescence imaging

ABSTRACT Traditional methods of localizing and quantifying the presence of pathogenic microorganisms in living experimental animal models of infections have mostly relied on sacrificing the animals, dissociating the tissue and counting the number of colony forming units. However, the discovery of several varieties of the light producing enzyme, luciferase, and the genetic engineering of bacteria, fungi, parasites and mice to make them emit light, either after administration of the luciferase substrate, or in the case of the bacterial lux operon without any exogenous substrate, has provided a new alternative. Dedicated bioluminescence imaging (BLI) cameras can record the light emitted from living animals in real time allowing non-invasive, longitudinal monitoring of the anatomical location and growth of infectious microorganisms as measured by strength of the BLI signal. BLI technology has been used to follow bacterial infections in traumatic skin wounds and burns, osteomyelitis, infections in intestines, Mycobacterial infections, otitis media, lung infections, biofilm and endodontic infections and meningitis. Fungi that have been engineered to be bioluminescent have been used to study infections caused by yeasts (Candida) and by filamentous fungi. Parasitic infections caused by malaria, Leishmania, trypanosomes and toxoplasma have all been monitored by BLI. Viruses such as vaccinia, herpes simplex, hepatitis B and C and influenza, have been studied using BLI. This rapidly growing technology is expected to continue to provide much useful information, while drastically reducing the numbers of animals needed in experimental studies.

DNA labeling in vivo: quantification of epidermal stem cell chromatin content in whole mouse hair follicles using Fiji image processing software.

DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cells (LRC) in long-term experiments. A major hindrance of this methodology relies on the selection of adequate procedures to quantify the nucleoside analogue content from image data files. Here we propose a simple procedure using Fiji image processing software to accurately calculate nucleoside analogue retaining chromatin/total chromatin (LRC/DAPI) signal ratios in the well-known mouse hair follicle stem cell niche.