Latchezar Avramov

Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal Gram-positive and Gram-negative pathogenic bacteria.

The Gram‐negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic‐resistant bacteria, antimicrobial Photodynamic therapy (aPDT) seems to be a plausible alternative. In this work, photosensitization was performed on Gram‐positive and Gram‐negative bacteria in pure culture using new‐age cationic porphyrins, namely mesoimidazolium‐substituted porphyrin derivative (ImP) and pyridinium‐substituted porphyrin derivative (PyP). The photophysical properties of both the sensitizers including absorption, fluorescence emission, quantum yields of the triplet excited states and singlet oxygen generation efficiencies were evaluated in the context of aPDT application. The studied porphyrins exhibited high ability to accumulate into bacterial cells with complete penetration into early stage biofilms. As compared with ImP, PyP was found to be more effective for photoinactivation of bacterial strains associated with periodontitis, without any signs of dark toxicity, owing to its high photocytotoxicity.

Long wavelength absorbing cationic Zn(II)-phthalocyanines as fluorescent contrast agents for B16 pigmented melanoma

Three cationic zinc phthalocyanines (ZnPcs), tetrakis-(3-methylpyridyloxy)-, tetrakis-(3-hexyl-pyridyloxy)-, and tetrakis-(3-dodecylpyridyloxy)phthalocyaninezinc (ZnPcMe, ZnPcHe and ZnPcDo) have been studied as advanced fluorescent contrast agents for pigmented melanoma tumor. UV-vis spectroscopic properties of the monomers were investigated. Their photophysical behavior as a substantial part of dye-induced fluorescence was evaluated. The selective accumulation and labeling capacity towards B16F0 pigmented melanoma tumor were determined. Melanin containing cells were isolated and incubated with ZnPcs at several time intervals (1, 1.5 and 6 h) following the kinetics of cellular uptake. The highest accumulation was found for ZnPcHe. A lower uptake was detected for the more lipophilic ZnPcDo and more hydrophilic ZnPcMe. The fluorescence diagnostic potential of ZnPcs towards pigmented melanoma by using an argon-dye laser detection set-up was demonstrated.

Investigation of relations between skin cancer lesions’ images and their fluorescent spectra

This investigation is based on images obtained from healthy tissue and skin cancer lesions and their fluorescent spectra of cutaneous lesions derived after optical stimulation. Our analyses show that the lesions’ spectra of are different of those, obtained from normal tissue and the differences depend on the type of cancer. We use a comparison between these “healthy” and “unhealthy” spectra to define forms of variations and corresponding diseases. However, the value of the emitted light varies not only between the patients, but also depending on the position of the tested area inside of one lesion. These variations could be result from two reasons: different degree of damaging and different thickness of the suspicious lesion area. Regarded to the visible image of the lesion, it could be connected with the chroma of colour of the tested area and the lesion homogeneity that corresponds to particular disease. For our investigation, images and spectra of three non-melanoma cutanous malignant tumors are investigated, namely—basal cell carcinoma, squamous cell carcinoma, and keratoacanthoma. The images were processed obtaining the chroma by elimination of the background—healthy tissue, and applying it as a basic signal for transformation from RGB to Lab colorimetric model. The chroma of the areas of emission is compared with the relative value of fluorescence spectra. Specific spectral features are used to develop hybrid diagnostic algorithm (including image and spectral features) for differentiation of these three kinds of malignant cutaneous pathologies.

5-ALA Mediated Fluorescence Detection of Gastrointestinal Tumors

Correspondence should be addressed to Ekaterina Borisova, borisova@ie.bas.bgReceived 7 March 2008; Revised 7 May 2008; Accepted 19 June 2008Recommended by Stoyan TanevDelta-aminolevulinic acid/protoporphyrin IX is applied for fluorescent tumor detection in the upper part of gastrointestinal tract.The 5-ALA is administered per os six hours before measurements at dose 20mg/kg weight. High-power light-emitting diodeat 405nm is used as an excitation source. Special opto-mechanical device is built to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence tomicrospectrometer. In such way, 1D detection and 2D visualization of the lesions’ fluorescence are received, and both advantagesand limitations of these methodologies are discussed in relation to their clinical applicability. Comparison of the spectra receivedfrom normal mucosa, inflammatory, and tumor areas is applied to evaluate the feasibility for development of simple but effectivealgorithm based on dimensionless ratio of the fluorescence signals at 560 and 635nm, for differentiation of normal/abnormalgastrointestinal tissues.Copyright

LIGHT-INDUCED FLUORESCENCE SPECTROSCOPY AND OPTICAL COHERENCE TOMOGRAPHY OF BASAL CELL CARCINOMA

Many up-to-date optical techniques have been developed and applied recently in clinical practice for obtaining qualitatively and quantitatively new data from the investigated lesions. Due to their high sensitivity in detection of small changes, these techniques are widely used for detection of early changes in biological tissues. Light-induced fluorescence spectroscopy (LIFS) is one of the most promising techniques for early detection of cutaneous neoplasia. Increasing number of recent publications have suggested that optical coherence tomography (OCT) also has potential for non-invasive diagnosis of skin cancer. This recent work is a part of clinical trial procedure for introduction of LIFS technique into the common medical practice in National Oncological Medical Center in Bulgaria for diagnosis of non-melanoma skin cancer. We focus our attention here on basal cell carcinoma lesions and their specific features revealed by LIFS and OCT analysis. In this paper we prove the efficiency of using the combined LIFS-OCT method in skin lesions studies by integrating the complimentary qualities of each particular technique. For LIFS measurements several excitation sources, each emitting at 365, 385 and 405 nm maxima are applied. An associated microspectrometer detects in vivo the fluorescence signals from human skin. The main spectral features of the lesions and normal skin are discussed and their possible origins are indicated. OCT images are used to evaluate the lesion thickness, structure and severity stage, when possible. The obtained results could be used to develop a more complete picture of optical properties of these widely spread skin disorders. At the same time, our studies show that the combined LIFS-OCT method could be introduced in clinical algorithms for early tumor detection and differentiation between normal/benign/malignant skin lesions.

The Stress and Vascular Catastrophes in Newborn Rats: Mechanisms Preceding and Accompanying the Brain Hemorrhages

In this study, we analyzed the time-depended scenario of stress response cascade preceding and accompanying brain hemorrhages in newborn rats using an interdisciplinary approach based on: a morphological analysis of brain tissues, coherent-domain optical technologies for visualization of the cerebral blood flow, monitoring of the cerebral oxygenation and the deformability of red blood cells (RBCs). Using a model of stress-induced brain hemorrhages (sound stress, 120 dB, 370 Hz), we studied changes in neonatal brain 2, 4, 6, 8 h after stress (the pre-hemorrhage, latent period) and 24 h after stress (the post-hemorrhage period). We found that latent period of brain hemorrhages is accompanied by gradual pathological changes in systemic, metabolic, and cellular levels of stress. The incidence of brain hemorrhages is characterized by a progression of these changes and the irreversible cell death in the brain areas involved in higher mental functions. These processes are realized via a time-depended reduction of cerebral venous blood flow and oxygenation that was accompanied by an increase in RBCs deformability. The significant depletion of the molecular layer of the prefrontal cortex and the pyramidal neurons, which are crucial for associative learning and attention, is developed as a consequence of homeostasis imbalance. Thus, stress-induced processes preceding and accompanying brain hemorrhages in neonatal period contribute to serious injuries of the brain blood circulation, cerebral metabolic activity and structural elements of cognitive function. These results are an informative platform for further studies of mechanisms underlying stress-induced brain hemorrhages during the first days of life that will improve the future generations health.

Light-induced autofluorescence and diffuse reflectance spectroscopy in clinical diagnosis of skin cancer

We investigated more than 500 clinical cases to receive the spectral properties of basal cell (136 patients) and squamous cell carcinoma (28), malignant melanoma (41) and different cutaneous dysplastic and benign cutaneous lesions. Excitation at 365, 385 and 405 nm using LEDs sources is applied to obtain autofluorescence spectra, and broad-band illumination in the region of 400-900 nm is used to detect diffuse reflectance spectra of all pathologies investigated. USB4000 microspectrometer (Ocean Optics Inc, USA) is applied as a detector and fiber-optic probe is used for delivery of the light. In the case of in vivo tumor measurements spectral shape and intensity changes are observed that are specific for a given type of lesion. Autofluorescence origins of the signals coming from skin tissues are mainly due to proteins, such as collagen, elastin, keratin, their cross-links, co-enzimes – NADH and flavins and endogenous porphyrins. Spectral features significant into diffuse spectroscopy diagnosis are related to the effects of re-absorption of hemoglobin and its forms, as well as melanin and its concentration in different pathologies. We developed significant database and revealed specific features for a large class of cutaneous neoplasia, using about 30 different spectral peculiarities to differentiate cutaneous tumors. Sensitivity and specificity obtained exceed 90%, which make optical biopsy very useful tool for clinical practice. These results are obtained in the frames of clinical investigations for development of significant “spectral features” database for the most common cutaneous malignant, dysplastic and benign lesions. In the forthcoming plans, our group tries to optimize the existing experimental system for optical biopsy of skin, and to introduce it and the diagnostic algorithms developed into clinical practice, based on the high diagnostic accuracy achieved.

Light-Induced Fluorescence Techniques for Gastrointestinal Tumour Detection

Gastrointestinal tumors have major place in the statistics of newly developed cancers every year, as the colon cancer is on third place, stomach cancer is on fifth place, and esophageal cancer is also in the top ten of tumors according statistics of cancer incidence. Usually the tumors are detected on advanced III and IV stage, where the perspectives for the patients are not very optimistic (Danon, 2003; Jemal, 2011). Up to now white light endoscopy is the main method in detection of gastrointestinal tumors. White-light endoscopy is wellestablished and wide used modality. However, despite the many technological advances that have been occurred, conventional white light endoscopy is suboptimal and usually detects lesions, which already have symptoms of obstruction, bleeding and pain, related to tumor growth. Misdiagnoses, related to difficulties in differentiation of inflammatory from initial stage adenocarcinoma also have negative effect on the diagnostic accuracy (Da Costa, 2003). Only experienced gastroenterologists with long practice in endoscopy observations could find slight initial changes to dysplastic and neoplastic stages of esophageal, stomach or colon mucosa. The limitations of standard endoscopy for detection and evaluation of cancerous changes in gastrointestinal tract are significant challenge and initiate development of new diagnostic modalities. Such detection and visualization techniques, additional to standard endoscopy equipment, including optical detection of tissues alterations are investigated and their feasibilities for clinical usage are evaluated. Advances in fiber optics, light sources, detectors have led to the development of several novel methods for tissue evaluation in situ. Optical methods applied for such tissue evaluation often are referred under term “optical biopsy”, which indicate their possibilities to make an instant diagnosis at endoscopy, previously possible only by using of histological and/or cytological analysis (Wang, 2004). The new optical approaches are based on light-tissue interactions and differences occurred between normal and abnormal tissue sites. In gastroenterology several optical methods are applied recently, such as optical coherent tomography (Tumlinson, 2004), chromo-endoscopy, confocal fluorescent microscopy (DaCosta, 2003a; 2003b), Raman

5-ALA/PpIX fluorescence detection of gastrointestinal neoplasia

In the recent study delta-ALA/PpIX is used as fluorescent marker for dysplasia and tumor detection in esophagus, stomach and colon. ALA is administered per os six to eight (depending on the lesion location) hours before measurements at dose 20mg/kg weight. High-power light-emitting diode at 405 nm is used as an excitation source. Special opto-mechanical device is built for the LED to use the light guide of standard video-endoscopic system. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. The fluorescence detected from tumor sites has very complex spectral origins. It consists of autofluorescence, fluorescence from exogenous fluorophores and re-absorption from the chromophores accumulated in the tissue investigated. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to re-absorption of oxy-hemoglobin in this spectral area. Endogenous and exogenous fluorescence spectra are used to develop simple but effective algorithm, based on dimensionless ratio of the signals at 560 and 635 nm, for differentiation of normal/abnormal gastrointestinal tissues. Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

Insulin potentiation of photodynamic activity in vitro

The present work reports on the investigation of the possible influence of insulin on the photodynamic effect of the photosensitizer radachlorin on four cell lines: HepG2, HT-29, BJ and BALB/3T3. For a quantitative comparison, the insulin effect on the standard chemotherapy drug methotrexate is also measured. The results show that the EC50 value for methotrexate is 40% higher than when combined with insulin; and for the lowest methotrexate concentration used, the difference exceeds 120%. For the case of the photodynamic effect, it is shown that insulin, when applied in combination with radachlorin, improves the cell permeability for the photosensitizer, thus increasing its concentration in the cells. The cytotoxic effect of photodynamic therapy with radachlorin as the photosensitizer has been quantitatively compared. The quantitative comparison of the photodynamic effect in the cases of using the photosensitizer radachlorin both alone and in combination with insulin, shows that the addition of insulin doe...