Photodynamic Therapy utilizing Interstitial Light Delivery Combined with Spectroscopic Methods

Since cancer continues to plague humanity there is large need for development of modalities for both diagnosis and therapy. Most of the currently available methods suffer from serious disadvantages. The treatments, e.g. ionising radiation, chemotherapy, surgery, may themselves induce malignancies or the patient may be physically impaired for a longer period of time. The work presented aims at developing equipment and methods that use light for both detection and treatment of various malignant or pre-malignant conditions. Fundamental knowledge on the interaction between light and tissue is required in order to develop models for the light distribution in tissue. Therefore, basic properties of light-tissue interaction, like refractive index, absorption, scattering, and scattering anisotropy, are introduced. How the physiological status of the tissue affects these properties are discussed. Utilizing the differences in the fluorescence spectra emitted by healthy and malignant tissues, when irradiated with visible light, it is possible to detect and delineate certain lesions. The contrast between diseased and healthy tissue can be further enhanced with the use of a fluorescence tumour marker. The evolution of these tumour markers has been fuelled by the fact that many tumour markers also can be utilized for light therapy. The modality is called photodynamic therapy (PDT) and has now been clinically approved for the treatment of several conditions. The possible indications for this type of treatment are generally limited to thin superficial lesions due to the limited penetration of the light in tissue. The work presented in this thesis mainly relates to overcoming the limited light penetration by leading the light through multiple optical fibres inserted into the tumour. In this way both embedded tumour and/or thick tumours could be an indication for this modality. In addition to that the fibres are used to collect information about relevant parameters of therapeutic interest

Clinical system for interstitial photodynamic therapy with combined on-line dosimetry measurements

A system for interstitial photodynamic therapy with delta-aminolaevulinic acid and multiple optical fibers has been developed. The system enables photodynamic treatment of large embedded tumor volumes and utilizes real-time measurements to allow on-line dosimetry. Important parameters such as light fluence rate, sensitizer fluorescence intensity, and changes in local blood oxygen saturation are measured with the same fibers that deliver the therapeutic light. Data from the first clinical treatments on nodular basal cell carcinomas indicate a major treatment-induced light absorption increase, rapid sensitizer photo-bleaching, and a relatively constant global tissue oxygen saturation level during the treatment

Feasibility study of a system for combined light dosimetry and interstitial photodynamic treatment of massive tumors

A system for the photodynamic laser treatment of massive tumors that employs multiple optical fibers to be inserted into the tumor mass is described. The light flux through the tumor can be assessed by use of the individual fibers both as transmitters and as receivers. With a computer model that describes the diffusive light propagation, optical dosimetry is under development, The system has been tested in an experimental animal tumor model in preparation for clinical work. Currently, delta-aminolevulinic acid is used as a sensitizer, activated by 635-nm radiation from a 2.0-W compact diode laser system. With the availability of future, highly selective drugs absorbing approximately 750 nm, larger tumor volumes should be treatable, and surrounding, sensitive normal tissue should be spared

Interstitial photodynamic therapy - diagnostic measurements and treatment in rat malignant experimental tumours

A recently developed multiple fibre system for treating malignant tumours with interstitial photodynamic therapy was used in studies on rats with colon adenocarcinoma inoculated into the muscles of the hind legs. The animals were intraperitonially administrated delta -aminolevulinic acid (ALA), which is metabolised to protoporphyrin IX (PpIX) in the tissue. The treatment system consists of a laser light source, a beam-splitting system dividing the light into three or six output fibres and a dosimetry programme calculating the optimal fibre position within the tumour as well as the treatment time needed to obtain a given threshold value of the light dose. One aim of the study was to compare the treatment outcome with the modelled dosimetry predictions. Tumour reduction was examined three days post treatment. A volume decrease was found in 85\% of the treated tumours. The mean volume reduction was 44\%, with one tumour completely disappearing. Histopathological examination three days post treatment showed substantial necrotic parts which, however, to a smaller extent were present also for non-treated tumours. These results indicated that the tumours have been under treated and the light dose has to be increased. Measurements of the build-up and photo-induced bleaching of PpIX using laser-induced fluorescence were also performed during the experiments

Analysis of spatial variability in hyperspectral imagery of the uterine cervix in vivo

The use of fluorescence and reflectance spectroscopy in the analysis of cervical histopathology is a growing field of research. The majority of this research is performed with point-like probes. Typically, clinicians select probe sites visually, collecting a handful of spectral samples. An exception to this methodology is the Hyperspectral Diagnostic Imaging (HSDI®) instrument developed by Science and Technology International. This non-invasive device collects contiguous hyperspectral images across the entire cervical portio. The high spatial and spectral resolution of the HSDI instruments make them uniquely well suited for addressing the issues of coupled spatial and spectral variability of tissues in vivo. Analysis of HSDI data indicates that tissue spectra vary from point to point, even within histopathologically homogeneous regions. This spectral variability exhibits both random and patterned components, implying that point monitoring may be susceptible to significant sources of noise and clutter inherent in the tissue. We have analyzed HSDI images from clinical CIN (cervical intraepithelial neoplasia) patients to quantify the spatial variability of fluorescence and reflectance spectra. This analysis shows the spatial structure of images to be fractal in nature, in both intensity and spectrum. These fractal tissue textures will limit the performance of any point-monitoring technology

A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Bothrops </it>is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of <it>Bothrops alternatus</it>, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay.</p> <p>Results</p> <p>A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A₂(5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A₂were essentially acidic; no basic PLA₂were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed.</p> <p>Conclusions</p> <p><it>Bothrops alternatus </it>venom gland contains the major toxin classes described for other <it>Bothrops </it>venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA₂agrees with the lower myotoxicity of this venom compared to other <it>Bothrops </it>species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.</p

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

In vivo measurement of parameters of dosimetric importance during interstitial photodynamic therapy of thick skin tumors

A system for interstitial photodynamic therapy is used in the treatment of thick skin tumors. The system allows simultaneous measurements of light fluence rate, sensitizer fluorescence, and tissue oxygen saturation by using the same fibers as for therapeutic light delivery. Results from ten tumor treatments using delta-aminolevulinic acid (ALA)-induced protoporphyrin IX show a significant, treatment-induced increase in tissue absorption at the therapeutic wavelength, and rapid sensitizer photobleaching. The changes in oxy- and deoxyhemoglobin content are monitored by means of near-infrared spectroscopy, revealing a varying tissue oxygenation and significant changes in blood volume during treatment. These changes are consistent with the temporal profiles of the light fluence rate at the therapeutic wavelength actually measured. We therefore propose the observed absorption increase to be due to treatment-induced deoxygenation in combination with changes in blood concentration within the treated volume. A higher rate of initial photobleaching is found to correlate with a less pronounced increase in tissue absorption. Based on the measured signals, we propose how real-time treatment supervision and feedback can be implemented. Simultaneous study of the fluence rate, sensitizer fluorescence, and local tissue oxygen saturation level may contribute to the understanding of the threshold dose for photodynamic therapy. (c) 2006 Society of Photo-Optical Instrumentation Engineers

Photodynamic therapy and diagnostic measurements of basal cell carcinomas using esterified and non-esterified delta-aminolevulinic acid

Various optical techniques were used to investigate relevant parameters involved in photodynamic therapy (PDT) of human basal cell carcinomas (BCCs). The aim of the study was to compare the diagnostic and therapeutic outcome when using topically applied methyl-esterified delta -aminolevulinic acid (ALA-ME) and delta -aminolevulinic acid (ALA). A total of 35 pathologically verified BCCs in 14 patients were investigated. A diode laser. emitting continuous light at 633 nm, was used to induce PDT. The diagnostic measurements were performed before, during, and after PDT. Laser-induced fluorescence (LIF) was used to monitor the build-up of the ALA/ALA-ME-induced protoporphyrin IX (PpIX), The superficial tissue perfusion was measured with laser-Doppler perfusion imaging (LDPI) and the temperature of the lesion and the surrounding tissue was imaged with an IR-camera. A clear demarcation between the lesion and the normal skin was detected with LIF before the treatment for both PpIX precursors. The fluorescence measurements suggest that PpIX builds up to a higher degree and more selectively in the tumour following ALA-ME as compared to ALA. The LDPI measurements indicate a local transient restriction in blood perfusion immediately post-PDT. The measurement with the IR-camera revealed a temperature rise of about 1-2 degreesC during the treatment