Hilde H. Buzzá

Experience and BCC subtypes as determinants of MAL-PDT response: Preliminary results of a national Brazilian project

Non-melanoma skin cancer is the most prevalent cancer type in Brazil and worldwide. Photodynamic therapy (PDT) is a noninvasive technique with excellent cosmetic outcome and good curative results, when used for the initial stages of skin cancer. A Brazilian program was established to determine the efficacy of methyl aminolevulinate (MAL)-PDT, using Brazilian device and drug. The equipment is a dual device that combines the photodiagnosis, based on widefield fluorescence, and the treatment at 630nm. A protocol was defined for the treatment of basal cell carcinoma with 20% MAL cream application. The program also involves the training of the medical teams at different Brazilian regions, and with distinct facilities and previous PDT education. In this report we present the partial results of 27 centers with 366 treated BCC lesions in 294 patients. A complete response (CR) was observed in 76.5% (280/366). The better response was observed for superficial BCC, with CR 160 lesions (80.4%), when compared with nodular or pigmented BCC. Experienced centers presented CR of 85.8% and 90.6% for superficial and nodular BCC respectively. A high influence of the previous doctor experience on the CR values was observed, especially due to a better tumor selection.

Evaluation of vascular effect of photodynamic therapy in chorioallantoic membrane using different photosensitizers

Photodynamic Therapy (PDT) is a local treatment that requires a photosensitizing agent, light and molecular oxygen. With appropriate illumination, the photosensitizer is excited and produces singlet oxygen that is highly reactive and cytotoxic. Tumor vascular network is essential for the tumor growth and the understanding of vascular response mechanisms enables an improvement in the PDT protocol for cancer treatment. Compounds of porphyrin (Photogem®) and chlorin (Photodithazine®) were the photosensitizers tested. The incubation times varied from 20 to 80 min and the concentration ranged between 0.1 and 100 μg/cm(2). Different light doses were used between 4.8 and 40 J/cm(2) with irradiance varying between 80 and 100 mW/cm(2). The light dose of 30 J/cm(2) was used in the intravenous photosensitizer application. The membrane images were made from 0 to 300 min after treatment. The vascular response was evaluated by the average vessel area. Different responses was observed depending on the photosensitizer concentration and administration form. Intravenous application has been more efficient to produce vessel constriction and the most pronounced effect was observed for the chlorin.

Photodynamic therapy: Progress toward a scientific and clinical network in Latin America.

Cancer is one of the major challenges for Latin America health services, since the skin cancer is the most frequent lesion. This manuscript addresses an initiative for the treatment of basal cell carcinomas (BCC) by photodynamic therapy (PDT) based on a government-funded national program in Brazil. The program provides clinical training and facilitates access to drugs/equipment and significantly reduces PDT costs. It also lays foundations for the establishment of a Latin American research network to improve prevention, early detection and treatment of diseases. Centers have been established by direct contact (conferences, visits to healthcare facilities and official departments). A local training was divided into complementary theoretical and practical parts. This is an ongoing project that has involved 10 countries: Brazil, Bolivia Chile, Ecuador, El Salvador, Colombia, Cuba, Mexico, Peru and Venezuela, The initial results are encouraging and have provided assessment of Latin America patients relating, for example, the most common skin phototypes with incidence of BCC in such countries. The network is expected to produce relevant scientific information for PDT introduction in many countries. The experience acquired by local teams shall enable them to innovate PDT protocols and increase the number of skilled contributors/researchers to broaden knowledge on the ever-crescent PDT field in Latin America. The establishment of a collaboration network and introduction of other projects and experience exchange shall become an easier process with time. This PDT clinical research network is a start for the strengthening of Science in South Hemisphere countries.

Single LED-based device to perform widefield fluorescence imaging and photodynamic therapy

Photodynamic therapy (PDT) is a treatment modality that can be indicated for several cancer types and pre-cancer lesions. One of the main applications of PDT is the treatment of superficial skin lesions such as basal cell carcinoma, Bowen’s disease and actinic keratosis. Three elements are necessary in PDT, a photosensitizer (PS); light at specific wavelength to be absorbed by the PS, and molecular oxygen. A typical PS used for skin lesion is protoporphyrin IX (PpIX), which is an intrinsic PS; its production is stimulated by a pro-drug, such as 5-aminolevulinic acid (ALA). Before starting a treatment, it is very important to follow up the PpIX production (to ensure that enough PS was produced prior to a PDT application) and, during a PDT session, to monitor its photodegradation (as it is evidence of the photodynamic effect taking place). The aim of this paper is to present a unique device, LINCE (MMOptics - São Carlos, Brazil), that brings together two probes that can, respectively, allow for fluorescence imaging and work as a light source for PDT treatment. The fluorescence probe of the system is optically based on 400 nm LED (light emitting diodes) arrays that allow observing the fluorescence emission over 450 nm. The PDT illumination probe options are constituted of 630 nm LED arrays for small areas and, for large areas, of both 630 nm and 450 nm LED arrays. Joining both functions at the same device makes PDT treatment simpler, properly monitorable and, hence, more clinically feasible. LINCE has been used in almost 1000 PDT treatments of superficial skin lesions in Brazil, with 88.4% of clearance of superficial BCC.

Evaluation of the Photodynamic Therapy effect using a tumor model in Chorioallantoic Membrane with Melanoma cells

Photodynamic Therapy (PDT) is a type of cancer treatment that is based on the interaction of light (with specific wavelength), a photosensitizing agent and molecular oxygen. The photosensitizer (PS) is activated by light and reacts with oxygen resulting in the production of singlet oxygen that is highly reactive and responsible for the cell death. The Chick Chorioallantoic Membrane (CAM) model is a transparent membrane that allows visualization and evaluation of blood vessels and structural changes, where a tumor model was developed. Two induction tumor models were investigated: tumor biopsy or cell culture. It was used a murine melanoma cell B16F10 in culture and a biopsy from a xenograft tumor in hairless mouse. Two PS were tested: Photodithazine® and Photogem®, a chlorine and porphyrin compounds, respectively. Using intravenous administration, the light-drug interval was of 30 minutes, 1 and 3 hours. Illumination was performed at 630 nm and 660 nm, and the vascular and tumor response was monitored and analyzed. The PS distribution was checked with confocal microscopy. This model can be useful to study several parameters of PDT and the effect of this therapy in the cancer treatment since it allows direct visualization of its effects.

Assembly and characterization of a nonlinear optical microscopy for in vivo and ex vivo tissue imaging

The purpose of this study is the assembly and characterization of a custom-made non-linear microscope. The microscope allows the adjustment for in vitro, in vivo and ex vivo imaging of biological samples. Two galvanometer mirrors conjugated by two spherical mirrors are used for the lateral scan and for the axial scan a piezoeletric stage is utilized. The excitation is done using a tunable femtosecond Ti: Sapphire laser. The light is focused in tissue by an objective lens 20X, water immersion, numerical aperture of 1.0, and working distance of 2.0 mm. The detection system is composed by a cut off filter that eliminates laser light back reflections and diverse dichroic filters can be chosen to split the emitted signal for the two photomultiplier detector. The calibration and resolution of the microscope was done using a stage micrometer with 10 μm divisions and fluorescent particle slide, respectively. Fluorescence and second harmonic generation images were performed using epithelial and hepatic tissue, the images have a sub-cellular spatial resolution. Further characterization and differentiation of tissue layers can be obtained by performing axial scanning. By means of the microscope it is possible to have a three dimensional reconstruction of tissues with sub-cellular resolution.

Photostimulation effects on chicken egg development: Perspectives on human newborn treatment

It is well known that, under exposure to bright light, eggs tend to hatch earlier than control, without any damage to the birds. This report aims to systematically show the effect and establishes a proposal for a possible application to accelerate chicken egg formation, which could be extrapolated or adapted as a great advance in premature human newborns. Comparing several protocols, the experiments show that lower doses of light slowly delivered for 24 h promote higher efficiency in embryo development, increasing on average 25% of its size and more than 70% in weight when compared to the control. This weight difference shows promising results compared to rates of up to 17% found in the literature. These results can be a first step to reduce the stay of premature human infants in hospitals because light, when applied in very low doses, can accelerate the natural biological processes without risks.

Fluorescence analysis of a tumor model in the chorioallantoic membrane used for the evaluation of different photosensitizers for photodynamic therapy

The development of a tumor in the chicken chorioallantoic membrane (CAM) enables a more individualized understanding of the dynamics of the photosensitizer (PS) interaction with the tumor blood vessels and cells. Photogem® and 5-aminolevulinic acid (ALA), a protoporphyrin IX (PpIX) precursor, were used as PS and their red fluorescence enabled the monitoring of PS dynamic distribution in the vessels and in the tumor. With a tumor model in CAM and fluorescence assessment, the aim of this study was to evaluate the PDT parameters comparing different photosensitezers. In this model, the topical application was chosen as the best way of drug delivery and widefield fluorescence images were at every 30min. The images were processed in a MATLAB® routine for a semi-quantitative analysis of the red fluorescence. PpIX formation in the blood vessels and in the tumor region was observed after 3h and 1.5h, respectively, whereas Photogem® was accumulated in the tumor region after 2h. The illumination was performed by a diode laser with emission centered at 635nm and irradiance of 80mW/cm2 for 10min. After PDT irradiation, the photobleaching for both compounds was observed. Photogem® showed a reduced photobleaching, however, both PS induced a destruction of the tumor mass and vascular network in the treated area.

Probing conformational changes in orphan nuclear receptor: The NGFI-B intermediate is a partially unfolded dimer

Human nerve growth factor-induced B (NGFI-B) is a member of the NR4A subfamily of orphan nuclear receptors (NRs). Lacking identified ligands, orphan NRs show particular co-regulator proteins binding properties, different from other NRs, and they might have a non-classical quaternary organization. A body of evidence suggests that NRs recognition of and binding to ligands, DNA, homo- and heterodimerization partners and co-regulator proteins involve significant conformational changes of the NR ligand-binding domains (LBDs). To shed light on largely unknown biophysical properties of NGFI-B, here we studied structural organization and unfolding properties of NGFI-B ligand (like)-binding domain induced by chemical perturbation. Our results show that NGFI-B LBD undergoes a two-state guanidine hydrochloride (GndHCl) induced denaturation, as judged by changes in the alpha-helical content of the protein monitored by circular dichroism spectroscopy (CD). In contrast, changes in the tertiary structure of NGFI-B LBD, reported by intrinsic fluorescence, reveal a clear intermediate state. Additionally, SAXS results demonstrate that the intermediate observed by intrinsic fluorescence is a partially folded homodimeric structure, which further unfolds without dissociation at higher GndHCl concentrations. This partially unfolded dimeric assembly of NGFI-B LBD might resemble an intermediate that this domain access momentarily in the native state upon interactions with functional partners.

Optical techniques for the diagnosis and treatment of lesions induced by the human papillomavirus — A resource letter

Human papillomaviruses (HPV) are the most common sexually-transmitted virus, and carcinogenic HPV strains are reported to be responsible for virtually all cases of cervical cancer and its precursor, the cervical intraepithelial neoplasia (CIN). About 30% of the sexually active population are considered to be affected by HPV. Around 600 million people are estimated to be infected worldwide. Diseases related to HPV cause significant impact from both the personal welfare point of view and public healthcare perspective. This resource letter collects relevant information regarding HPV-induced lesions and discusses both diagnosis and treatment, with particular attention to optical techniques and the challenges involved to the implementation of those approaches.