This PDF file contains the front matter associated with SPIE Proceedings Volume 6438, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Photodynamic therapy (PDT) is clinically established modality used for treatment of solid cancers and other conditions, which destroys lesions by localized generation of cytotoxic oxygen species mediated by administered drugs (photosensitizers) that are activated at targeted sites by exposure to light. Since over 20 years ago it has become increasingly clear that important contribution to the antitumor effect of PDT is secured by host reaction induced by this therapy and manifested as inflammatory and immune response. Presented is an overview of advances in the understanding of this host response associated with tumor PDT by tracing its evolution from initial breakthroughs and discoveries in the early 1980s, followed by advances preceding recent developments, and concluding with recently acquired knowledge and directions for clinical exploitation. Tribute is given to researchers making important contributions to this field during the last three decades including Drs. Gianfranco Canti, Julia Levy, and Barbara Henderson.

Cancer is a leading cause of death among modern peoples largely due to metastatic disease. The ideal cancer treatment should target both the primary tumor and the metastases with the minimal toxicity. This is best accomplished by educating the body's immune system to recognize the tumor as foreign so that after the primary tumor is destroyed, distant metastases will also be eradicated. Photodynamic therapy (PDT) involves the IV administration of photosensitizers followed by illumination of the primary tumor with red light producing reactive oxygen species that cause vascular shutdown and tumor cell apoptosis. Anti-tumor immunity is stimulated after PDT due to the acute inflammatory response, priming of the immune system to recognize tumor-associated antigens (TAA), and induction of heat-shock proteins. The induction of specific CD8+ T lymphocyte cells that recognize major histocompatibility complex class I (MHC-I) restricted epitopes of TAAs is a highly desirable goal in cancer therapy. We here report on PDT of mice bearing tumors that either do or do not express an established TAA. We utilized a BALB/c colon adenocarcinoma cell line termed CT26.CL25 retrovirally transduced to stably express &bgr;-galactosidase ( &bgr;-gal, a bacterial protein), and its non-&bgr;-gal expressing wild-type counterpart termed CT26 WT, as well as the control cell line consisting of CT26 transduced with the empty retroviral vector termed CT26-neo. All cells expressed class I MHC restriction element H-2Ld syngenic to BALB/c mice. Vascular PDT with a regimen of 1mg/kg BPD injected IV, and 120 J/cm² of 690-nm laser light after 15 minutes successfully cured 100% of CT26.CL25 tumors but 0% of CT26-neo tumors and 0% of CT26 WT tumors. After 90 days tumor free interval the CT26.CL25 cured mice were rechallenged with CT26.CL25 tumor cells and 96% rejected the rechallenge while the CT26.CL25 cured mice did not reject a CT26 WT tumor cell challenge. Experiments with mice bearing two CT26.CL25 tumors (one in each leg) and only one tumor treated with PDT, showed that the immune response was strong enough to destroy an already established tumor in 70 of the mice and this effect was not seen with mice bearing two CT26 WT tumors. We expect these studies will lead to an understanding of the relevant determinants of immune response after PDT that could be rapidly applied to patient-selection and improvement in outcome for PDT for cancer.

In situ Photoimmunotherapy (ISPI) was developed to treat metastatic tumors using a combination of phototherapy and immunotherapy. It utilizes local intervention through photothermal destruction of existing solid tumors and through immune response modifier to elicit host anti-tumor responses. Such combination in pre-clinical studies has shown promise in cancer treatment by eradicating the primary tumors and also controlling metastases at distant sites. ISPI has been used in our preliminary clinical studies for melanoma patients and the outcome has been extremely encouraging. In 2006, we began enrolling patients in a new phase I immunotherapy trial for advanced cutaneous melanoma. This trial is based on our previous results which indicated that we had developed an effective treatment for advanced melanoma. Of the first six patients treated, (4 stage IV, and 2 surgically unresectable stage III), 2 of the stage IV patients are still alive, one tumor free, and one with a possible treatable recurrence after 2 1/2 years. We have also discovered that recurrences of the skin cancer can be retreated by the same technique and that treatment seems to blunt the virulence of the disease and make it more treatable. These initial results indicate that ISPI probably will have the ability to prolong survival in selected cases of advanced melanoma, and potentially cure a significant percentage of treated patients.

To investigate the feasibility and efficacy of combination of imiquimod immunotherapy and 5- aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) for the treatment of genital bowenoid papulosis (BP). A total of twenty seven BP patients were randomized into two groups: (I) fifteen patients (12 male and 3 female, age 22-56 years old) were treated with topical application of 5% imiquimod cream (three times a week) and ALA-PDT (100 J/cm² at 100 mW/cm², once a week) for 1-4 times in one week interval. (II) Twelve patients (6 male and 6 female, age 29-58 years old) were treated with CO₂ laser vaporization as a control. Patients were followed up for 3 to 12 months. Results: In combined therapy group, 60% (9/15) patients showed complete remission and only one recurred (11.1%) during follow up. Local side effects included mild erythema, edema, erosion and burning and/or stinging sensation. No systemic side effect was found. In CO₂ laser vaporization group, 83.3% (10/12) patients showed complete remission. However, recurrence occurred in 6 patients (60.0%). Local side effects included mild to moderate edema, erosion, ulceration, delayed healing, prolonged pain and scarring. The difference of recurrence rate between two groups was statistically significant (P < 0.05). Topical application of imiquimod cream and ALA-PDT is safe, effective and associated with low recurrence and less side effect. Its true clinical value needs to be further investigated by a long-term follow-up of large scale trial.

Objectives: To investigate the pharmacokinetics of ALA induced protoporphyrin IX (PpIX) in lesions of urethral condylomata acuminata. Methods: Sixty patients (20 - 60 years old, 48 male and 12 female) with urethral condylomata acuminata were divided randomly into 5 groups to receive different concentrations of ALA solution (0.5, 1, 3, 5 or 10%). The ALA solution was applied topically to the lesion for a different length of time (1, 3, 5 or 7 h). Biopsy was performed at the end of incubation and specimens were subjected to histological and PpIX fluorescence analyses. Results: ALA-induced PpIX fluorescence was dominantly distributed in the epidermis. The maximal fluorescence intensity was detected at the 5 h of incubation. Higher ALA concentration (e.g. 5 and 10%) produced stronger intensity. In contrast, only the minimal amount of PpIX fluorescence was detected in the dermis. Conclusions: The results suggest that the topical application of 5 - 10% ALA solution for 3-5 h are the optimal conditions for ALA/PpIX-mediated photodynamic therapy for the treatment of urethral condylomata acuminata.

Immune system is critical in the fight against cancer. Particular important is the responses through immune cells that regulate immunological functions. Certain cytokines enhance cancer immunity (such as IL12 and interferon gamma) and others interfere or impede cancer immunity (such as IL10). The clinical outcome can be linked to the balance of these cytokines, such as IL10 to IL12 ratio. Effective treatments often reduce the IL10:IL12 ratio, indicating higher levels of the cancer fighting IL12. To enhance immune responses, a combination of laser irradiation and concurrent use of immunostimulants has been applied for the treatment of tumors. In a recent study, an 805-nm laser in conjunction with indocyanine green (ICG) has been used to treat EMT6 mammary tumors in mice. An immunoadjuvant, glycated chitosan (GC), was intratumoral injected after the laser irradiation. Our preliminary results showed that tumor-bearing mice treated either with the immunoadjuvant alone or with the combination of laser and immunoadjuvant had lower IL10:IL12 ratios than animals that received no treatment. This may play an important in the treatment to decrease tumor size and to increase survival times of mice. Cellular activities after laser-ICG-GC treatment of DBMA-4 mammary tumors in rats also showed infiltration of immune cells to the treatment sites, indicating a possible induced immunity. The combination of laser treatment and immunotherapy has been used to treat late-stage melanoma patients; the responses, both treated primary tumors and the metastases, to the treatment have been promising. The histology of two patients, before and after treatment, is presented to show the effects of this novel treatment method.

In cancer treatment and immune response enhancement research, Magnetic Resonance Imaging (MRI) is an ideal method for non-invasive, three-dimensional temperature measurement. We used a 7.1-Tesla magnetic resonance imager for ex vivo tissues and small animal to determine temperature distribution of target tissue during laser irradiation. The feasibility of imaging is approved with high spatial resolution and high signal-noise- ratio. Tissue-simulating gel phantom gel, biological tissues, and tumor-bearing animals were used in the experiments for laser treatment and MR imaging. Thermal couple measurement of temperature in target samples was used for system calibration. An 805-nm laser was used to irradiate the samples with a laser power in the range of 1 to 2.5 watts. Using the MRI system and a specially developed processing algorithm, a clear temperature distribution matrix in the target tissue and surrounding tissue was obtained. The temperature profiles show that the selective laser photothermal effect could result in tissue temperature elevation in a range of 10 to 45 °C. The temperature resolution of the measurement was about 0.37°C including the total system error. The spatial resolution was 0.4 mm (128x128 pixels with field of view of 5.5x5.5 cm). The temperature distribution provided in vivo thermal information and future reference for optimizing dye concentration and irradiation parameters to achieve optimal thermal effects in cancer treatment.

An ideal cancer treatment method should not only cause primary tumor suppression but also induce an antitumor immunity, which is essential for control of metastatic tumors. A combination therapy using a laser, a laser-absorbing dye, and an immunoadjuvant guided by temperature measurement probes such as magnetic resonance imaging thermometry (MRT) and infrared thermography (IRT) can be an ideal treatment modality. Temperature distribution inside the target tissue is important in laser treatment. The surface temperature often serves as an indicator of the treatment effect. However, real-time monitoring of surface temperature during laser irradiation poses a great challenge. In this study, we investigated the surface temperature distribution using direct measurement and theoretical simulation. The preliminary results of in vitro and in vivo studies are presented. Gel phantom and chicken breast tissue were irradiated by an 805 nm laser and the surface temperature distribution was obtained using an infrared thermal camera. EMT-6 breast tumors in mice were treated using the 805 nm laser and with different dye and immunoadjuvant combinations, including intratumor injections of indocyanine green (ICG) and glycated chitosan (GC). Monte Carlo simulation for selective photothermal-tissue interaction was also performed for the surface temperature distributions. Our results demonstrated that the tissue temperature can be accurately monitored in real time and can be controlled by appropriate treatment parameters.

Low-power laser irradiation (LPLI) has been shown to promote cell proliferation in various cell types, yet the mechanism of which has not been fully clarified. Investigating the signaling pathways involved in the laser irradiation is important for understanding these processes. The small G protein Ras works as a binary switch in many important intracellular signaling pathways and, therefore, has been one of the focal targets of signal-transduction investigations and drug development. The Ras/Raf/MEK/ERK (extracellular-signal-regulated kinase) signaling pathway is a network that governs proliferation, differentiation and cell survival. Recent studies suggest that Ras/Raf signaling pathway is involved in the LPLI-induced cell proliferation. On the other hand, Protein kinase Cs (PKCs), the Ca²⁺ activated, phospholipid-dependent serine/threonine protein kinases, have been recently presumed to be involved in the regulation of cell proliferation induced by LPLI. In this report, to monitor the direct activations of Ras and PKCs after LPLI treatment in living cells in real time, Raichu-Ras reporter and C kinase activity reporter (CKAR) were utilized, both of which were constructed based on fluorescence resonance energy transfer (FRET) technique. The direct activation of Ras is predominantly initiated from the different microdomains of the plasma membrane. The results are monitored during cell proliferation induced by LPLI (0.8 J/cm²) in serum-starved COS-7 cells expressing Raichu-Ras reporter using FRET imaging on laser scanning confocal microscope. Furthermore, the increasing activation of PKCs is also monitored during cell proliferation induced by LPLI (0.8 J/cm²) in serum-starved human lung adenocarcinoma cells (ASTC-a-1) expressing CKAR reporter using the similar way. Taken together, the dynamic increases of H-Ras and PKCs activities are observed during the processes of cell proliferation induced by LPLI.

Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways. Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during cisplatin-induced apoptosis.

Objectives: To study the cytotoxicity induced by photosensitizer mediated sonodynamic effects on c6 glioma cells in vitro and to lay the experimental foundation for the sonodynamic therapy (SDT) of glioma tumor. Methods: A domestic photosensitizer hematoporphyrin monomethyl ether (HMME) was used as the sensitizer. The growth inhibition rate of c6 glioma cells at different times after SDT was investigated by MTT assay. The apoptotic rate and cell circle profiles of c6 glioma cells was examined by a flow cytometry. Results: The growth inhibition rate of c6 glioma cells after SDT significantly increased. Ultrasound only group also showed some effects. Whereas, the inhibition rate of HMME only group has no significant difference compared with the control group (without ultrasound and HMME). HMME-mediated SDT increased the apoptotic rate and the percentage of S phase cells but decreased the percentages of G0/G1 and G2/M. SDT also increased the apoptosis and proliferation rate (APR). Conclusions: Our preliminary data indicated that SDT could kill c6 glioma cells in vitro and possibility through induction of apoptosis and arrest cell circle at S/G₂.

Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction.^1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

Many cellular events involve the alteration in redox equilibrium, globally or locally. In many cases, excessive reactive oxygen species (ROS) production is the underlying cause. Several green fluoresecence protein based indicators are constructed to measure redox status in cells, e.g, rxYFP and roGFPs, which allow real time detection. reduction and oxidization-sensitive GFP (RoGFPs) are more useful due to ratiometric variation by excitation, making the measurement more accurate. Utilizing one of those roGFPs called roGFP1, we establish a mitochondrial redox state probing platform in HeLa cells with laser scan confocal microscopy (LSCM) as detection system. Control experiments confirmed that our platform could produce stable ratiometric values, which made the data more accurately reflect the real environmental changes of redox status that roGFP1 probed. Using exogenous H₂O₂ and DTT, we evaluated the reactivity and reversibility of roGFP1. The minimal hydrogen peroxide concentration that roGFP1 could show detectable ratiometric changes in our system was about 200&mgr;M. Preliminarily applying our platform to exploring the redox status during apoptosis, we observed an increase in ratiometric, suggesting an excessive ROS production.

Time-resolved photoacoustic spectroscopy is a novel and potential tool for the noninvasive measurements of chromophore concentrations in vivo. In this study, noninvasive measurement of concentration and oxygen saturation of hemoglobin has been investigated by using photoacoustic method. We detailedly report a home-made photoacoustic experiment system for this study. In our system, a Q-switched ND: YAG pulse laser operating at 1064nm with a 10ns pulse width has been employed to generate photoacoustic signals. The photoacoustic signals, generated by varying the hemoglobin concentration or oxygenation saturation in blood experimentally, were picked up and analyzed. The results show that the photoacoustic technique is a useful and helpful tool for noninvasive monitoring of the total hemoglobin concentration and the oxygen saturation, for it can accurately detect the variation of the total hemoglobin concentration and oxygen saturation of hemoglobin, even when the blood vessel is deep in high scattering medium for 1cm.

Photodynamic therapy (PDT) is a method to treat cancer or non-cancer diseases by activation of the light-sensitive photosensitizers. Epstein Barr virus (EBV) has been implicated in the development of certain cancers such as nasopharyngeal carcinoma and B cell lymphoma. This study aims to examine the effects of EBV infection on the production of pro-inflammatory cytokines and chemokines in cells after the photosensitizer Zn-BC-AM PDT treatment. Epithelial tumor cell lines HONE-1 and latent EBV-infected HONE-1 (EBV-HONE-1) cells were used in this study. Cells were treated with the photosensitizer Zn-BC-AM for 24 hours before light irradiation. RT-PCR and quantitative ELISA methods were used for the evaluation of mRNA expression and production of cytokines, respectively. Results show that Zn-BC-AM PDT increases the production of IL-1a and IL-1b in EBV-HONE-1. Over a 10-fold increase in the production of IL-6 was observed in the culture supernatant of Zn-BC-AM PDT-treated HONE-1 cells. PDT-induced IL-6 production was observed in HONE-1 cells. EBV-HONE-1 has a higher background level of IL-8 production than the HONE-1. The production of IL-8 was suppressed in EBV-HONE-1cells after Zn-BC-AM PDT. Our results indicate that the response of HONE-1 cells to Zn-BC-AM PDT depends on the presence of latent EBV infection. Since IL-8 is a cytokine with angiogenic activity, Zn-BC-AM PDT may exert an anti-angiogenic effect through the suppression of IL-8 production by the EBV-infected cells.

Immune state monitoring is an expensive, invasive and sometimes difficult necessity in patients with different disorders. Immune reaction dynamics study in patients with coronary atherosclerosis provides one of the leading components to complication development, clinical course prognosis and treatment and rehabilitation tactics. We've chosen intravenous glucose injection as metabolic irritant in the following four groups of patients: men with proved coronary atherosclerosis (CA), non insulin dependent diabetes mellitus (NIDDM), men hereditary burden by CA and NIDDM and practically healthy persons with longlivers in generation. Immune state parameters such as quantity of leukocytes and lymphocytes, circulating immune complexes levels, serum immunoglobulin levels, HLA antigen markers were studied at 0, 30 and 60 minutes during glucose loading. To obtain continues time function of studied parameters received data were approximated by polynomials of high degree with after going first derivatives. Time functions analyze elucidate principally different dynamics studied parameters in all chosen groups of patients, which couldn't be obtained from discontinuous data compare. Leukocyte and lymphocyte levels dynamics correlated HLA antigen markers in all studied groups. Analytical estimation of immune state in patients with coronary atherosclerosis shows the functional "margin of safety" of immune system state under glucose disturbance. Proposed method of analytical estimation also can be used in immune system monitoring in other groups of patients.