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Purpose: For interstitial photodynamic therapy (iPDT) of bulky tumors, careful treatment planning is required in order to ensure that a therapeutic dose is delivered to the tumor, while minimizing damage to surrounding normal tissue. In clinical contexts, iPDT has typically been performed with either flat cleaved or cylindrical diffusing optical fibers as light sources. Here, the authors directly compare these two source geometries in terms of the number of fibers and duration of treatment required to deliver a prescribed light dose to a tumor volume. Methods: Treatment planning software for iPDT was developed based on graphics processing unit enhanced Monte Carlo simulations. This software was used to optimize the number of fibers, total energy delivered by each fiber, and the position of individual fibers in order to deliver a target light dose (D90) to 90% of the tumor volume. Treatment plans were developed using both flat cleaved and cylindrical diffusing fibers, based on tissue volumes derived from CT data from a head and neck cancer patient. Plans were created for four cases: fixed energy per fiber, fixed number of fibers, and in cases where both or neither of these factors were fixed. Results: When the number of source fibers was fixed at eight, treatment plans based on flat cleaved fibers required each to deliver 7180-8080 J in order to deposit 90 J/cm2 in 90% of the tumor volume. For diffusers, each fiber was required to deliver 2270-2350 J (333-1178 J/cm) in order to achieve this same result. For the case of fibers delivering a fixed 900 J, 13 diffusers or 19 flat cleaved fibers at a spacing of 1 cm were required to deliver the desired dose. With energy per fiber fixed at 2400 J and the number of fibers fixed at eight, diffuser fibers delivered the desired dose to 93% of the tumor volume, while flat cleaved fibers delivered this dose to 79%. With both energy and number of fibers allowed to vary, six diffusers delivering 3485-3600 J were required, compared to ten flat cleaved fibers delivering 2780-3600 J. Conclusions: For the same number of fibers, cylindrical diffusers allow for a shorter treatment duration compared to flat cleaved fibers. For the same energy delivered per fiber, diffusers allow for the insertion of fewer fibers in order to deliver the same light dose to a target volume. [ABSTRACT FROM AUTHOR]

We examined effects of fluence rate on the photobleaching of the photosensitizer Pc 4 during photodynamic therapy (PDT) and the relationship between photobleaching and tumor response to PDT. BALB/c mice with intradermal EMT6 tumors were given 0.03 mg kg−1 Pc 4 by intratumor injection and irradiated at 667 nm with an irradiance of 50 or 150 mW cm−2 to a fluence of 100 J cm−2. While no cures were attained, significant tumor growth delay was demonstrated at both irradiances compared with drug-only controls. There was no significant difference in tumor responses to these two irradiances ( P = 0.857). Fluorescence spectroscopy was used to monitor the bleaching of Pc 4 during irradiation, with more rapid bleaching with respect to fluence shown at the higher irradiance. No significant correlation was found between fluorescence photobleaching and tumor regrowth for the data interpreted as a whole. Within each treatment group, weak associations between photobleaching and outcome were observed. In the 50 mW cm−2 group, enhanced photobleaching was associated with prolonged growth delay ( P = 0.188), while at 150 mW cm−2 this trend was reversed ( P = 0.308). Thus, it appears that Pc 4 photobleaching is not a strong predictor of individual tumor response to Pc 4-PDT under these treatment conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT Cutaneous and mucocutaneous Candida infections are considered to be important targets for antimicrobial photodynamic therapy (PDT). Clinical application of antimicrobial PDT will require strategies that enhance microbial killing while minimizing damage to host tissue. Increasing the sensitivity of infectious agents to PDT will help achieve this goal. Our previous studies demonstrated that raising the level of oxidative stress in Candida by interfering with fungal respiration increased the efficiency of PDT. Therefore, we sought to identify compounds in clinical use that would augment the oxidative stress caused by PDT by contributing to reactive oxygen species (ROS) formation themselves. Based on the ability of the antifungal miconazole to induce ROS in Candida, we tested several azole antifungals for their ability to augment PDT in vitro. Although miconazole and ketoconazole both stimulated ROS production in Candida albicans, only miconazole enhanced the killing of C. albicans and induced prolonged fungistasis in organisms that survived PDT using the porphyrin TMP-1363 and the phenothiazine methylene blue as photosensitizers. The data suggest that miconazole could be used to increase the efficacy of PDT against C. albicans, and its mechanism of action is likely to be multifactorial. [ABSTRACT FROM AUTHOR]

Mucosal infections caused by the pathogenic fungus Candida are a significant infectious disease problem and are often difficult to eradicate because of the high frequency of resistance to conventional antifungal agents. Photodynamic treatment (PDT) offers an attractive therapeutic alternative. Previous studies demonstrated that filamentous forms and biofilms of Candida albicans were sensitive to PDT using Photofrin as a photosensitizer. However, early stationary phase yeast forms of C. albicans and Candida glabrata were not adversely affected by treatment. We report that the cationic porphyrin photosensitizer meso-tetra ( N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) is effective in PDT against yeast forms of C. albicans and C. glabrata. Respiratory-deficient (RD) strains of C. albicans and C. glabrata display a pleiotropic resistance pattern, including resistance to members of the azole family of antifungals, the salivary antimicrobial peptides histatins and other types of toxic stresses. In contrast to this pattern, RD mutants of both C. albicans and C. glabrata were significantly more sensitive to PDT compared to parental strains. These data suggest that intact mitochondrial function may provide a basal level of anti-oxidant defense against PDT-induced phototoxicity in Candida, and reveals pathways of resistance to oxidative stress that can potentially be targeted to increase the efficacy of PDT against this pathogenic fungus. [ABSTRACT FROM AUTHOR]

We present an in vivo fluorescence microscopic evaluation of intratumor distribution of the photosensitizer mono-L-aspartylchlorin-e6 (NPe6) in an intradermal mouse EMT6 tumor model. Although the identification of favorable photophysical and pharmacological properties has led to the development of new photosensitizers in photodynamic therapy, their intratumor distribution kinetics have remained relatively understudied. In this study, we used confocal fluorescence microscopy to follow the transport of NPe6 in vivo after systemic administration through the tail vein. Labeling of vasculature using fluorophore-conjugated anti-CD31 antibodies allows visualization of the uptake of NPe6 in tumor and normal vessels and its partitioning kinetics into the adjacent parenchyma for 3 hours after injection. During the initial 60 minutes after injection, the drug is predominantly confined to the vasculature. Subsequently, it significantly redistributes throughout the extravascular regions with no discernable difference in its extravasation rate between tumor and normal tissues. Further, we investigate the sensitizer's altered intratumor distribution in response to photodynamic therapy irradiation and observe that treatment-induced changes in vessel permeability caused enhanced accumulation of NPe6 in the extravascular space. Our findings are of immediate clinical relevance and demonstrate the importance of an in vivo imaging approach to examine the dynamic process of intratumor drug distribution. [ABSTRACT FROM AUTHOR]

Genetically-encoded photosensitizers produce reactive oxygen species (ROS) in response to light. Transgenic expression of fusion proteins can target the photosensitizers to specific cell regions and permit the spatial and temporal control of ROS production. These ROS-generating proteins (RGPs) are widely used for cell ablation, mutagenesis and chromophore-assisted light inactivation of target proteins. However, the species produced by RGPs are unclear due to indirect measures with confounding interpretations. Recently, the RGP mini “Singlet Oxygen Generator” (miniSOG) was engineered from Arabidopsis thaliana phototropin 2. While miniSOG produces singlet oxygen ( 1 O 2 ), the contribution of superoxide (O 2 • - ) to miniSOG-generated ROS remains unclear. We measured the light-dependent O 2 • - production of purified miniSOG using HPLC separation of dihydroethidium (DHE) oxidation products. We demonstrate that DHE is insensitive to 1 O 2 and establish that DHE is a suitable indicator to measure O 2 • - production in a system that produces both 1 O 2 and O 2 • - . We report that miniSOG produces both 1 O 2 and O 2 • - , as can its free chromophore, flavin mononucleotide. miniSOG produced O 2 • - at a rate of ~4.0 µmol O 2 • - /min/µmol photosensitizer for an excitation fluence rate of 5.9 mW/mm 2 at 470 ± 20 nm, and the rate remained consistent across fluences (light doses). Overall, the contribution of O 2 • - to miniSOG phenotypes should be considered. [ABSTRACT FROM AUTHOR]

A correction to the article "Respiratory Deficiency Enhances the Sensitivity Candida to Photodynamic Treatment," by Yeissa Chabrier-Rossello and colleagues that was published in the 2008 issue is presented.

The article discusses various reports within the issue, including preclinical papers on potential cell death mechanisms and pathways and studies on photodynamic therapy.

We demonstrate the use of an enzyme-activatable fluorogenic probe, Neutrophil Elastase 680 FAST (NE680), for in vivo imaging of neutrophil elastase (NE) activity in tumors subjected to photodynamic therapy (PDT). NE protease activity was assayed in SCC VII and EMT6 tumors established in C3H and BALB/c mice, respectively. Four nanomoles of NE680 was injected intravenously immediately following PDT irradiation. 5 h following administration of NE680, whole-mouse fluorescence imaging was performed. At this time point, levels of NE680 fluorescence were at least threefold greater in irradiated versus unirradiated SCC VII and EMT6 tumors sensitized with Photofrin. To compare possible photosensitizer-specific differences in therapy-induced elastase activity, EMT6 tumors were also subjected to 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-PDT. NE levels measured in HPPH-PDT-treated tumors were twofold higher than in unirradiated controls. Ex vivo labeling of host cells using fluorophore-conjugated antibodies and confocal imaging were used to visualize Gr1+ cells in Photofrin-PDT-treated EMT6 tumors. These data were compared with recently reported analysis of Gr1+ cell accumulation in EMT6 tumors subjected to HPPH-PDT. The population density of infiltrating Gr1+ cells in treated versus unirradiated drug-only control tumors suggests that the differential in NE680 fold enhancement observed in Photofrin versus HPPH treatment may be attributed to the significantly increased inflammatory response induced by Photofrin-PDT. The in vivo imaging of NE680, which is a fluorescent reporter of NE extracellular release caused by neutrophil activation, demonstrates that PDT results in increased NE levels in treated tumors, and the accumulation of the cleaved probe tracks qualitatively with the intratumor Gr1+ cell population. [ABSTRACT FROM AUTHOR]

Meso-tetra-hydroxyphenyl-chlorin (mTHPC, Foscan®), a promising photosensitizer for photodynamic therapy (PDT), is approved in Europe for the palliative treatment of head and neck cancer. Based on work in mice that investigated optimal tumor accumulation, clinical protocols with Foscan® typically employ an interval of 96 h between systemic sensitizer administration and irradiation. However, recent studies in mouse tumor models have demonstrated significantly improved long-term tumor response when irradiation is performed at shorter drug-light intervals of 3 and 6 h. Using a previously published theoretical model of microscopic PDT dosimetry and informed by experimentally determined photophysical properties and intratumor sensitizer concentrations and distributions, we calculated photodynamic dose depositions following mTHPC-PDT for drug-light intervals of 3, 6, 24, and 96 h. Our results demonstrate that the singlet oxygen dose to the tumor volume does not track even qualitatively with tumor responses for these four drug-light intervals. Further, microscopic analysis of simulated singlet oxygen deposition shows that in no case do any subpopulations of tumor cells receive a threshold dose. Indeed, under the conditions of these simulations more than 90% of the tumor volume receives a dose that is approximately 20-fold lower than the threshold dose for mTHPC. Thus, in this evaluation of mTHPC-PDT at various drug-light intervals, any PDT dose metric that is proportional to singlet oxygen creation and/or deposition would fail to predict the tumor response. In situations like this one, other reporters of biological response to therapy would be necessary. [ABSTRACT FROM AUTHOR]

lntracellular calcium concentration ([Ca2+]i) signals are central to the mechanisms underlying fluid and protein secretion in pancreatic and parotid acinar cells. Calcium release was studied in natively buffered cells following focal laser photolysis of caged molecules. Focal photolysis of caged-inositol 1,4,5 trisphosphate (InsP3) in the apical region resulted in Ca2+ release from the apical trigger zone and, after a latent period, the initiation of an apical-to-basal Ca2+ wave. The latency was longer and the wave speed significantly slower in pancreatic compared with parotid cells. Focal photolysis in basal regions evoked only limited Ca2+ release at the photolysis site and never resulted in a propagating wave. Instead, an apical-to-basal wave was initiated following a latent period. Again, the latent period was significantly longer under all conditions in pancreas than parotid. Although slower in pancreas than parotid, once initiated, the apical-to-basal wave speed was constant in a particular cell type. Photo release of caged-Ca2+ failed to evoke a propagating Ca2+ wave in either cell type. However, the kinetics of the Ca2+ signal evoked following photolysis of caged-InsP3 were significantly dampened by ryanodine in parotid but not pancreas, indicating a more prominent functional role for ryanodine receptor (RyR) following InsP3 receptor (InsP3R) activation. These data suggest that differing expression levels of InsP3R, RyR, and possibly cellular buffering capacity may contribute to the fast kinetics of Ca2+ signals in parotid compared with pancreas. These properties may represent a specialization of the cell type to effectively stimulate Ca2+-dependent effectors important for the differing primary physiological role of each gland. [ABSTRACT FROM AUTHOR]

We have developed a comprehensive theoretical model for rigorously describing the spatial and temporal dynamics of oxygen (3O2) consumption and transport and microscopic photodynamic dose deposition during photodynamic therapy (PDT) in vivo. Previously published models have been improved by considering perfused vessels as a time-dependent 3O2 source and linking the 3O2 concentration in the vessel to that within the tissue through the Hill equation. The time-dependent photochemical 3O2 consumption rate incorporates sensitizer photobleaching effects and an experimentally determined initially nonuniform photosensitizer distribution. The axial transport of 3O2 is provided for in the capillaries and in the surrounding tissue. A self-sensitized singlet oxygen (1O2)-mediated bleaching mechanism and the measured, initially nonuniform distribution of meso-tetrahydroxyphenyl chlorin at 3 h after intravascular administration were used to demonstrate the capabilities of the model. Time-evolved distributions of 3O2 concentration were obtained by numerically solving two-dimensional diffusion-with-reaction equations both in the capillary and the adjacent tissue. Using experimentally established physiological and photophysical parameters, the mathematical model allows computation of the dynamic variation of hemoglobin-3O2 saturation (SO2) within the vessels, irreversible sensitizer degradation due to photobleaching, and the microscopic distributions of 3O2, sensitizer concentration, and 1O2 dose deposition under various irradiation conditions. The simulations reveal severe axial gradients in 3O2 and in photodynamic dose deposition in response to a wide range of clinically relevant treatment parameters. Thus, unlike former Krogh cylinder-based models, which assume a constant 3O2 concentration at the vessel, this new model identifies conditions in which 3O2 depletion and minimal deposition of reacting 1O2 exist near the end of axial segments of vessels and shows that treatment-limiting 3O2 depletion is induced at fluence rates as low as 10 mW cm-2. These calculations also demonstrate that intercapillary heterogeneity of photosensitizer contributes significantly to the distribution of photodynamic dose. This more rigorous mathematical model enables comparison with experimentally observable, volume-averaged quantities such as SO2 and the loss of sensitizer fluorescence through bleaching that have not been included in previous analyses. Further, it establishes some of the intrinsic limitations of such measurements. Specifically, our simulations demonstrate that tissue measurements of SO2 and of photobleaching are necessarily insensitive to microscopic heterogeneity of photodynamic dose deposition and are sensitive to intercapillary spacing. Because prior knowledge of intercapillary distances in tumors is generally unavailable, these measurements must be interpreted with caution. We anticipate that this model will make useful dosimetry predictions that should inform optimal treatment conditions and improve current clinical protocols. [ABSTRACT FROM AUTHOR]

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O 2 •-) and singlet oxygen (1O 2) quantum yields with irradiation at 561 nm. The formation of the O 2 •--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O 2 •- quantum yield (ΦO 2 •-) of SuperNova was determined to be 1.5 × 10-3, KillerRed was 0.97 × 10-3, and mCherry 1.2 × 10-3. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 μM O 2 •-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O 2 quantum yield (Φ1O 2) for SuperNova to be 22.0 × 10-3, KillerRed 7.6 × 10-3, and mCherry 5.7 × 10-3. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology. Image 1 • We report O 2 •- and 1O 2 quantum yields for KillerRed, SuperNova and mCherry. • O 2 •- generation was measured using HPLC separation of 2-OHE+ and 1O 2 with SOSG. • Supernova's O 2 •- and 1O 2 yields are larger than those of KillerRed and mCherry. • O 2 •- quantum yield of mCherry is comparable to those of KillerRed and SuperNova. [ABSTRACT FROM AUTHOR]

A correction to the article "A comprehensive mathematical model of microscopic dose deposition in photodynamic therapy" that was published in the 2007 issue is presented.

Aims: How mitochondrial reactive oxygen species (ROS) impact physiological function may depend on the quantity of ROS generated or removed, and the subcellular microdomain in which this occurs. However, pharmacological tools currently available to alter ROS production in vivo lack precise spatial and temporal control. Results: We used CRISPR/Cas9 to fuse the light-sensitive ROS-generating protein, SuperNova to the C-terminus of mitochondrial complex II succinate dehydrogenase subunits B (SDHB-1::SuperNova) and C (SDHC-1::SuperNova) in Caenorhabditis elegans to localize SuperNova to the matrix-side of the inner mitochondrial membrane, and to the intermembrane space (IMS), respectively. The presence of the SuperNova protein did not impact complex II activity, mitochondrial respiration, or C. elegans development rate under dark conditions. ROS production by SuperNova protein in vitro in the form of superoxide (O2˙−) was both specific and proportional to total light irradiance in the 540–590 nm spectra, and was unaffected by varying the buffer pH to resemble the mitochondrial matrix or IMS environments. We then determined using SuperNova whether stoichiometric ROS generation in the mitochondrial matrix or IMS had distinct effects on redox signaling in vivo. Phosphorylation of PMK-1 (a p38 MAPK homolog) and transcriptional activity of SKN-1 (an Nrf2 homolog) were each dependent on both the site and duration of ROS production, with matrix-generated ROS having more prominent effects. Furthermore, matrix- but not IMS-generated ROS attenuated susceptibility to simulated ischemia reperfusion injury in C. elegans. Innovation and Conclusion: Overall, these data demonstrate that the physiological output of ROS depends on the microdomain in which it is produced. Antioxid. Redox Signal. 31, 594–607. [ABSTRACT FROM AUTHOR]

Abstract Oxidants play an important role in the cell and are involved in many redox processes. Oxidant concentrations are maintained through coordinated production and removal systems. The dysregulation of oxidant homeostasis is a hallmark of many disease pathologies. The local oxidant microdomain is crucial for the initiation of many redox signaling events; however, methods to control oxidant product are limited. Some fluorescent proteins, including GFP, TagRFP, KillerRed, miniSOG, and their derivatives, generate oxidants in response to light. These genetically-encoded photosensitizers produce singlet oxygen and superoxide upon illumination and offer spatial and temporal control over oxidant production. In this review, we will examine the photosensitization properties of fluorescent proteins and their application to redox biology. Emerging concepts of selective oxidant species production via photosensitization and the impact of light on biological systems are discussed. Graphical abstract fx1 Highlights • Some fluorescent proteins generate large quantities of oxidants upon illumination. • Genetically-encoded photosensitizers produce singlet oxygen and superoxide. • The contribution of each oxidant species for an application is often overlooked. • Advancements and limitations to these tools are highlighted. [ABSTRACT FROM AUTHOR]

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1 or TSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation of Tsc2 gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouse Tsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results in Tsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed in Tsc2-null myometrial tumors in an estrogen-dependent fashion. In vivo fluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterine Tsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated in Tsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers. [ABSTRACT FROM AUTHOR]

BACKGROUND Topical photodynamic therapy (PDT) for selected nonmelanoma skin cancer using 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) has yielded high long-term complete response rates with very good cosmesis. Pain during light activation of the photosensitizer can be a serious adverse event. A 2-step irradiance protocol has previously been shown to minimize ALA-PDT pain. OBJECTIVE To determine the irradiance-dependent pain threshold for MAL-PDT, to adapt the 2-step protocol to a light-emitting diode (LED) light source, and assess clinical response. METHODS In this prospective study, 25 superficial basal cell carcinoma (sBCC) received an initial irradiance by laser at 40 or 50 mW/cm², or LED at 35 mW/cm² followed by an irradiance at 70 mW/cm² for a total of 75 J/cm². Pain levels were recorded for both irradiance steps. Efficacy was assessed at 6, 12, or 24 months. RESULTS Pain was mild in the 40/70 mW/cm² laser cohort. Three instances of irradiance-limiting pain occurred at 50/70 mW/cm². Pain was minimal in the 35/70 mW/cm² LED cohort. Clinical response rates were 80% in the 50/70 mW/cm² laser cohort and 90% in the 35/70 mW/cm² LED cohort. CONCLUSION Topical PDT can be effectively delivered to sBCC with minimal treatment-related pain by a 2-step irradiance protocol. [ABSTRACT FROM AUTHOR]

Oxygen transport imposes a possible constraint on the brain's ability to sustain variable metabolic demands, but oxygen diffusion in the cerebral cortex has not yet been observed directly. We show that concurrent two-photon fluorescence imaging of endogenous nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation exposes well-defined boundaries of tissue oxygen diffusion in the mouse cortex. The NADH fluorescence increases rapidly over a narrow, very low pO2 range with a p50 of 3.4±0.6 mm Hg, thereby establishing a nearly binary reporter of significant, metabolically limiting hypoxia. The transient cortical tissue boundaries of NADH fluorescence exhibit remarkably delineated geometrical patterns, which define the limits of tissue oxygen diffusion from the cortical microcirculation and bear a striking resemblance to the ideal Krogh tissue cylinder. The visualization of microvessels and their regional contribution to oxygen delivery establishes penetrating arterioles as major oxygen sources in addition to the capillary network and confirms the existence of cortical oxygen fields with steep microregional oxygen gradients. Thus, two-photon NADH imaging can be applied to expose vascular supply regions and to localize functionally relevant microregional cortical hypoxia with micrometer spatial resolution. [ABSTRACT FROM AUTHOR]

Purpose: The present study investigates the efficacy of compartmental targeting in xenografted tumors treated by meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT). The therapeutic efficacy was, furthermore, related to a regional photoinduced distribution of apoptosis and an mTHPC biodistribution profile.Methods and Materials: Mice bearing EMT6 tumors were subjected to a single irradiation (10 J/cm(2)) of red laser light (652 nm) at different intervals after a single- (0.3 mg/kg or 0.15 mg/kg) or double-intravenous (2 × 0.15 mg/kg) injection(s) of mTHPC. Efficiency of the treatment was evaluated by monitoring tumor regrowth. mTHPC pharmacokinetics were assessed by high-performance liquid chromatography analysis of excised organs. The regional distribution of apoptosis in tumor sections was investigated with a newly developed colabelling immunohistochemistry technique.Results: A fractionated double-injection protocol of mTHPC with 24-h and 3-h drug-light intervals (DLI) yielded 100% tumor cure, with tumors presenting a massive apoptosis of neoplastic cells along with a distortion of vessels. The best efficiency for a single injection (0.3 mg/kg) was about 54% tumor cure and corresponded to a DLI of 3 h. At this DLI, tumors showed apoptosis of endothelial cells in residual vessels. Concentrations of mTHPC observed in plasma and tumor for the fractionated injection were not statistically different and were less than the total drug dose in each compartment.Conclusions: The present work suggests that clinical PDT protocols with mTHPC could be greatly improved by fractionation of the drug administration. Time points should be chosen based on the intratumoral spatiotemporal drug distribution. [ABSTRACT FROM AUTHOR]

Purpose: The present study investigates the efficacy of compartmental targeting in xenografted tumors treated by meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy (PDT). The therapeutic efficacy was, furthermore, related to a regional photoinduced distribution of apoptosis and an mTHPC biodistribution profile. Methods and Materials: Mice bearing EMT6 tumors were subjected to a single irradiation (10 J/cm2) of red laser light (652 nm) at different intervals after a single- (0.3 mg/kg or 0.15 mg/kg) or double-intravenous (2 × 0.15 mg/kg) injection(s) of mTHPC. Efficiency of the treatment was evaluated by monitoring tumor regrowth. mTHPC pharmacokinetics were assessed by high-performance liquid chromatography analysis of excised organs. The regional distribution of apoptosis in tumor sections was investigated with a newly developed colabelling immunohistochemistry technique. Results: A fractionated double-injection protocol of mTHPC with 24-h and 3-h drug-light intervals (DLI) yielded 100% tumor cure, with tumors presenting a massive apoptosis of neoplastic cells along with a distortion of vessels. The best efficiency for a single injection (0.3 mg/kg) was about 54% tumor cure and corresponded to a DLI of 3 h. At this DLI, tumors showed apoptosis of endothelial cells in residual vessels. Concentrations of mTHPC observed in plasma and tumor for the fractionated injection were not statistically different and were less than the total drug dose in each compartment. Conclusions: The present work suggests that clinical PDT protocols with mTHPC could be greatly improved by fractionation of the drug administration. Time points should be chosen based on the intratumoral spatiotemporal drug distribution. [Copyright &y& Elsevier]

Abstract: Respiratory deficiency increases the sensitivity of the pathogenic fungi Candida albicans and Candida glabrata to oxidative stress induced by photodynamic therapy (PDT) sensitized by the cationic porphyrin meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363). Since disruption of electron transport chain (ETC) function increases intracellular levels of reactive oxygen species in yeast, we determined whether interference with ETC assembly or function increased sensitivity to TMP-1363-PDT in C. albicans, C. glabrata and the non-pathogenic yeast Saccharomyces cerevisiae. Metabolic inhibitor antimycin A and defined genetic mutants were used to identify ETC components that contribute to the sensitivity to PDT. Inhibition of cytochrome bc1 (Complex III) with antimycin A increases mitochondrial levels of reactive oxygen species. PDT performed following pre-treatment with antimycin A reduced colony forming units (CFU) of C. albicans and C. glabrata by approximately two orders of magnitude relative to PDT alone. A S. cerevisiae mitochondrial glutaredoxin grx5 mutant, defective in assembly of Fe–S clusters critical for Complex III function, displayed increased sensitivity to PDT. Furthermore, C. glabrata and S. cerevisiae mutants in cytochrome c oxidase (Complex IV) synthesis and assembly were also significantly more sensitive to PDT. These included suv3, encoding an ATP-dependent RNA helicase critical for maturation of cytochrome c oxidase subunit transcripts, and pet117, encoding an essential cytochrome c oxidase assembly factor. Following PDT, the reduction in CFU of these mutants was one to two orders of magnitude greater than in their respective parental strains. The data demonstrate that selective inhibition of ETC Complexes III and IV significantly increases the sensitivity of C. albicans, C. glabrata and S. cerevisiae to PDT sensitized with TMP-1363. [Copyright &y& Elsevier]

The AKT/mammalian target of rapamycin (AKT/mTOR) and ERK MAPK signaling pathways have been shown to cooperate in prostate cancer progression and the transition to androgen-independent disease. We have now tested the effects of combinatorial inhibition of these pathways on prostate tumorigenicity by performing preclinical studies using a genetically engineered mouse model of prostate cancer. We report here that combination therapy using rapamycin, an inhibitor of mTOR, and PD0325901, an inhibitor of MAPK kinase 1 (MEK; the kinase directly upstream of ERK), inhibited cell growth in cultured prostate cancer cell lines and tumor growth particularly for androgen-independent prostate tumors in the mouse model. We further showed that such inhibition leads to inhibition of proliferation and upregulated expression of the apoptotic regulator Bcl-2-interacting mediator of cell death (Bim). Furthermore, analyses of human prostate cancer tissue microarrays demonstrated that AKT/mTOR and ERK MAPK signaling pathways are often coordinately deregulated during prostate cancer progression in humans. We therefore propose that combination therapy targeting AKT/mTOR and ERK MAPK signaling pathways may be an effective treatment for patients with advanced prostate cancer, in particular those with hormone-refractory disease. [ABSTRACT FROM AUTHOR]

Phthalocyanine 4 (Pc 4) is a promising photosensitizer currently in clinical trials. Photobiological responses to Pc 4 photodynamic therapy (Pc 4-PDT) have been characterized extensively, but relatively little has been done to evaluate dose metrics for this sensitizer. We describe an irradiation-induced increase in fluorescence in tumor cell monolayers. This increase is due solely to enhanced fluorescence from Pc 4, as confirmed by confocal spectroscopy. In EMT6 cells incubated with 250 n M Pc 4 for 24 h, the maximum increase in fluorescence is approximately 3.7-fold above baseline levels. This increase occurs over a range of fluences, 0.05 – 0.6 J cm−2, where clonogenic survival decreases by 3 orders of magnitude. Light scattering measurements performed on similarly treated EMT6 cells in suspension suggested a Pc 4-PDT-mediated mitochondrial swelling of approximately 13% at 0.6 J cm−2, where fluorescence enhancement saturates under these treatment conditions. Fluorescence imaging and light scattering experiments performed at a five-fold lower Pc 4 incubation concentration revealed a reduced fluorescence enhancement at a five-fold higher fluence, which produced comparable mitochondrial swelling. Taken together, these data suggest that Pc 4 is initially aggregated at high local concentration in mitochondria and that irradiation relaxes the quenching of Pc 4 fluorescence through a mechanism that may involve mitochondrial swelling. [ABSTRACT FROM AUTHOR]

We describe a technique for imaging enzyme activity through steady-state fluorescence anisotropy measurements on a per-pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy-FL-labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five-fold. Fluorescence lifetime and time-resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy-FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy-containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs. [ABSTRACT FROM AUTHOR]

We describe the design and implementation of an inverted laser scanning confocal fluorescence microscope utilizing the commercial Nikon Diaphot TMD platform. An external confocal scanner was retrofitted through the video side port of the Diaphot. With 10×, 0.5 NA dry and 60×, 1.4 NA oil immersion objectives, the depth discrimination is 5.8 μm and 0.8 μm, respectively, as determined by derivatives of fluorescence edge responses measured in liquid samples of rhodamine 6G dissolved in DMSO. We present sample edge response curves and representative confocal fluorescence images of tumor cells in monolayer culture. © 2001 American Institute of Physics. [ABSTRACT FROM AUTHOR]