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Biomedical subjects

Charles P Lin

Publications and source records attributed to Charles P Lin.

13 recordsLinked to original sources
PubMed2007

Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in multiple myeloma.

The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the P13K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

Animals↗
PubMed2006

G-CSF induces E-selectin ligand expression on human myeloid cells.

Clinical use of G-CSF can result in vascular and inflammatory complications. To investigate the molecular basis of these effects, we analyzed the adherence of G-CSF-mobilized human peripheral blood leukocytes (ML) to inflamed (TNF-alpha-stimulated) vascular endothelium. Studies using parallel plate assays under physiologic flow conditions and intravital microscopy in a mouse inflammation model each showed that ML take part in heightened adhesive interactions with endothelium compared to unmobilized (native) blood leukocytes, mediated by markedly increased E-selectin receptor-ligand interactions. Biochemical studies showed that ML express the potent E-selectin ligand HCELL (ref. 8) and another, previously unrecognized approximately 65-kDa E-selectin ligand, and possess enhanced levels of transcripts encoding glycosyltransferases (ST3GalIV, FucT-IV and FucT-VII) conferring glycan modifications associated with E-selectin ligand activity. Enzymatic treatments and physiologic binding assays showed that HCELL and the approximately 65-kDa E-selectin ligand contribute prominently to the observed G-CSF-induced myeloid cell adhesion to inflamed endothelium. Treatment of normal human bone marrow cells with a pharmacokinetically relevant concentration of G-CSF in vitro resulted in increased expression of these two molecules, coincident with increased transcripts encoding pertinent glycosyltransferases and heightened E-selectin binding. These findings provide direct evidence for a role of G-CSF in the induction of E-selectin ligands on myeloid cells, thus providing mechanistic insight into the pathobiology of G-CSF complications.

Animals↗
PubMed2005

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH(2) stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

Animals↗
PubMed2005

In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment.

The organization of cellular niches is known to have a key role in regulating normal stem cell differentiation and regeneration, but relatively little is known about the architecture of microenvironments that support malignant metastasis. Using dynamic in vivo confocal imaging, here we show that murine bone marrow contains unique anatomic regions defined by specialized endothelium. This vasculature expresses the adhesion molecule E-selectin and the chemoattractant stromal-cell-derived factor 1 (SDF-1) in discrete, discontinuous areas that influence the homing of a variety of tumour cell lines. Disruption of the interactions between SDF-1 and its receptor CXCR4 inhibits the homing of Nalm-6 cells (an acute lymphoblastic leukaemia cell line) to these vessels. Further studies revealed that circulating leukaemic cells can engraft around these vessels, suggesting that this molecularly distinct vasculature demarcates a microenvironment for early metastatic tumour spread in bone marrow. Finally, purified haematopoietic stem/progenitor cells and lymphocytes also localize to the same microdomains, indicating that this vasculature might also function in benign states to demarcate specific portals for the entry of cells into the marrow space. Specialized vascular structures therefore appear to delineate a microenvironment with unique physiology that can be exploited by circulating malignant cells.

Animals↗
PubMed2004

In vivo flow cytometry: a new method for enumerating circulating cancer cells.

The fate of circulating tumor cells is an important determinant of their ability to form distant metastasis. Here, we demonstrate the use of in vivo flow cytometry as a powerful new method for detecting quantitatively circulating cancer cells. We specifically examine the circulation kinetics of two prostate cancer cell lines with different metastatic potential in mice and rats. We find that the cell line and the host environment affect the circulation kinetics of prostate cancer cells, with the intrinsic cell line properties determining the initial rate of cell depletion from the circulation and the host affecting cell circulation at later time points.

Animals↗
PubMed2003

Targeting of the retinal pigment epithelium (RPE) by means of a rapidly scanned continuous wave (CW) laser beam.

BACKGROUND AND OBJECTIVES: Selective treatment of the retinal pigment epithelium (RPE) by repetitively applying green micros-laser pulses is a new method for retinal diseases associated with a degradation of the RPE, which spares the neural retina. We investigated an alternative approach to realize repetitive micros-laser exposure by rapidly scanning a continuous wave (CW)-laser beam across the RPE. STUDY DESIGN/MATERIALS AND METHODS: An Ar(+) laser beam (514 nm) with a diameter of 18.75 microm was repetitively scanned across porcine RPE samples in vitro providing an irradiation time of 1.6 micros per point on the central scan axis. RPE cell damage was investigated by means of the fluorescence viability assay Calcein-AM. RESULTS: The ED(50) cell damage is 305 mJ/cm(2) when applying 10 scans with a repetition rate of 500 Hz. The threshold decreases with the number of scans, a saturation was found at 135 mJ/cm(2) with more than 500 exposures applied. The depth of focus in beam direction is 350 microm, defined by an increase of the threshold radiant exposure by 20%. CONCLUSIONS: Targeting of pigmented cells with high local resolution has been proved with a laser-scanning device. Looking ahead selective RPE-treatment, the adaptation of a laser-scanning device on a slit-lamp or into a modified retina angiograph seems to be an attractive alternative to the pulsed micros laser device.

Animals↗
PubMed2003

Selective cell targeting with light-absorbing microparticles and nanoparticles.

We describe a new method for selective cell targeting based on the use of light-absorbing microparticles and nanoparticles that are heated by short laser pulses to create highly localized cell damage. The method is closely related to chromophore-assisted laser inactivation and photodynamic therapy, but is driven solely by light absorption, without the need for photochemical intermediates (particularly singlet oxygen). The mechanism of light-particle interaction was investigated by nanosecond time-resolved microscopy and by thermal modeling. The extent of light-induced damage was investigated by cell lethality, by cell membrane permeability, and by protein inactivation. Strong particle size dependence was found for these interactions. A technique based on light to target endogenous particles is already being exploited to treat pigmented cells in dermatology and ophthalmology. With exogenous particles, phamacokinetics and biodistribution studies are needed before the method can be evaluated against photodynamic therapy for cancer treatment. However, particles are unique, unlike photosensitizers, in that they can remain stable and inert in cells for extended periods. Thus they may be particularly useful for prelabeling cells in engineered tissue before implantation. Subsequent irradiation with laser pulses will allow control of the implanted cells (inactivation or modulation) in a noninvasive manner.

Apoptosis↗
PubMed2003

Selective uptake of indocyanine green by reticulocytes in circulation.

PURPOSE: Hyperfluorescent cells labeled with indocyanine green (ICG) have been observed in retinal and choroidal circulation using scanning laser ophthalmoscopy. It has been suggested that ICG labels leukocytes and that ICG can be used to track leukocyte movement in vivo. The purpose of this study is to identify the cell population that takes up ICG and to study their trafficking pattern in vivo by confocal fluorescence microscopy. METHODS: ICG was injected into the mouse tail vein, and images were taken by in vivo confocal microscopy. The trafficking pattern of ICG-labeled cells was compared with that of rhodamine 6G-labeled leukocytes. In vitro labeling of human blood cells with antibodies against cell lineage markers and with DNA stains was further used to identify the ICG-labeled cells. Antibodies against the following cell surface markers were used: CD45 (leukocytes), CD3 (T lymphocytes), CD19 (B lymphocytes), CD16 (Fc receptor), glycophorin A (erythroid lineage cells), and CD71 (transferrin receptor). RESULTS: The ICG-labeled cells were made up of two blood cell populations with distinct levels of ICG uptake. The strongly ICG-labeled cells did not roll on dermal vascular endothelium in vivo, in contrast to rhodamine 6G-labeled leukocytes. They were identified as reticulocytes because antibody staining showed that they were CD 45(-), glycophorin A(+) and CD 71(+). The weakly ICG-labeled cells were identified as neutrophils because they were CD45(+), CD16(+), CD3(-), and CD19(-). CONCLUSIONS: ICG strongly labels reticulocytes and weakly labels neutrophils. To the authors' knowledge, this is the first report of selective staining of reticulocytes by ICG.

Animals↗
PubMedDate not supplied

Cellular activation of the self-quenched fluorescent reporter probe in tumor microenvironment.

The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 microm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFP-negative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFP-negative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45- and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

Animals↗
PubMedDate not supplied

Pump-probe detection of laser-induced microbubble formation in retinal pigment epithelium cells.

Microsecond laser pulses are currently being investigated in a new ophthalmic procedure for treatment of disorders associated with the retinal pigment epithelium (RPE). The precise mechanism for microsecond laser-induced RPE damage, however, has not been determined. We have previously shown that short pulse laser irradiation in the nanosecond to picosecond time domain causes transient microbubble formation around melanin granules in pigmented cells. Nanosecond time-resolved microscopy was previously used to visualize the intracellular cavitation dynamics. However, this technique is difficult to use with microsecond laser exposures, especially when multiple laser pulses are applied in a rapid sequence as in the clinical setting. Here we describe a simple pump-probe method for detecting transient light scattering signal from individual RPE cells when they are irradiated with nanosecond and microsecond laser pulses. For single 12 ns pulses the threshold for bubble detection was the same as the ED(50) threshold for cell death. For 6 micros pulse duration the threshold for bubble detection was about 10% higher than the threshold for cell death. With repetitive pulse trains at 500 Hz the ED(50) decreased about 25% for 10 and 100 pulses. Cells die when a single bubble was detected in a multiple pulse sequence.

Air↗
PubMedDate not supplied

Selective targeting of the retinal pigment epithelium using an acousto-optic laser scanner.

Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE.

Acoustics↗
PubMedDate not supplied

Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy.

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.

Animals↗