Modulating Expression of Endogenous Interleukin 1 Beta in the Acute Phase of the Pilocarpine Model of Epilepsy May Change Animal Survival.

The pilocarpine-induced (PILO) model has helped elucidate the electrophysiological and molecular aspects related to mesial temporal lobe epilepsy. It has been suggested that the extensive cell death and edema observed in the brains of these animals could be induced by increased inflammatory responses, such as the rapid release of the inflammatory cytokine interleukin 1 beta (Il1b). In this study, we investigate the role of endogenous Il1b in the acute phase of the PILO model. Our aim is twofold. First, we want to determine whether it is feasible to silence Il1b in the central nervous system using a non-invasive procedure. Second, we aim to investigate the effect of silencing endogenous Il1b and its antagonist, Il1rn.We used RNA interference applied non-invasively to knockdown Il1b and its endogenous antagonist Il1rn. We found that knocking down Il1b prior to pilocarpine injection increased the mortality rate of treated animals. Furthermore, we observed that, when exposing the animals to more Il1b by silencing its endogenous antagonist Il1rn, there was a better response to status epilepticus with decreased animal mortality in the acute phase of the PILO model. Thus, we show the feasibility of using a novel, less invasive approach to study genes involved in the inflammatory response in the central nervous system. Furthermore, our results provide suggestive evidence that modulating endogenous Il1b improves animal survival in the acute phase of the PILO model and may have effects that extend into the chronic phase.

Detection limits of multi-spectral optical imaging under the skin surface.

The present work shows that the optical/biological information contained in a typical spectral image mainly reflects the properties of a small (conic like) volume of tissue situated vertically under each individual pixel. The objects appearing on a spectral image reasonably reproduce the correct geometrical shape and size (like a non-deformed shadow) of underlying inclusions of pathological tissue. The information contained in a spectral image comes from a depth that does not exceed approximately 2-3 mm. The number of photons that visit a given tissue voxel situated at a depth larger than approximately 2 mm represents less than the 1% of the total number of photons reaching the corresponding detection pixel (forming the image). A pathological inclusion (e.g. a pool of blood or vascular tumor) situated at a depth of approximately 0.5 mm with a thickness of 0.5 mm produces an image intensity contrast of approximately 5% (for images taken at wavelengths in the 600-1000 nm range) when compared to the normal skin background. The same inclusion at a depth of 20 microm provides a contrast decreasing from 55 to 20% with respect to an increase in wavelength. The dermis/hypodermis interface behaves as a partial barrier for the photons, limiting their access to deeper skin regions. The image contrast depends on the depth and the type of chromophore contained in the inclusion. An increase in the concentration of a given molecule may produce different contrast, independently of the depth, depending on the characteristics of the skin layer where this change occurs.

Skin and cutaneous melanocytic lesion simulation in biomedical optics with multilayered phantoms.

The complex inner layered structure of skin influences the photon diffusion inside the cutaneous tissues and determines the reflectance spectra formation. Phantoms are very useful tools to understand the biophysical meaning of parameters involved in light propagation through the skin. To simulate the skin reflectance spectrum, we realized a multilayered skin-like phantom and a multilayered skin phantom with a melanoma-like phantom embedded inside. Materials used were Al(2)O(3) particles, melanin of sepia officinalis and a calibrator for haematology systems dispersed in transparent silicon. Components were optically characterized with indirect techniques. Reflectance phantom spectra were compared with average values of in vivo spectra acquired on a sample of 573 voluntary subjects and 132 pigmented lesions. The phantoms' reflectance spectra agreed with those measured in vivo, mimicking the optical behaviour of the human skin. Further, the phantoms were optically stable and easily manageable, and represented a valid resource in spectra formation comprehension, in diagnostic laser applications and simulation model implementation, such as the Monte Carlo code for non-homogeneous media.

Multispectral imaging and artificial neural network: mimicking the management decision of the clinician facing pigmented skin lesions.

Various instruments based on acquisition and elaboration of images of pigmented skin lesions have been developed in an attempt to in vivo establish whether a lesion is a melanoma or not. Although encouraging, the response of these instruments, e.g. epiluminescence microscopy, reflectance spectrophotometry and fluorescence imaging, cannot currently replace the well-established diagnostic procedures. However, in place of the approach to instrumentally assess the diagnosis of the lesion, recent studies suggest that instruments should rather reproduce the assessment by an expert clinician of whether a lesion has to be excised or not. The aim of this study was to evaluate the performance of a spectrophotometric system to mimic such a decision. The study involved 1794 consecutively recruited patients with 1966 doubtful cutaneous pigmented lesions excised for histopathological diagnosis and 348 patients with 1940 non-excised lesions because clinically reassuring. Images of all these lesions were acquired in vivo with a multispectral imaging system. The data set was randomly divided into a train (802 reassuring and 1003 excision-needing lesions, including 139 melanomas), a verify (464 reassuring and 439 excision-needing lesions, including 72 melanomas) and a test set (674 reassuring and 524 excision-needing lesions, including 76 melanomas). An artificial neural network (ANN(1)) was set up to perform the classification of the lesions as excision-needing or reassuring, according to the expert clinicians' decision on how to manage each examined lesion. In the independent test set, the system was able to emulate the clinicians with a sensitivity of 88% and a specificity of 80%. Of the 462 correctly classified as excision-needing lesions, 72 (95%) were melanomas. No major variations in receiver operating characteristic curves were found between the test and the train/verify sets. On the same data set, a further artificial neural network (ANN(2)) was then architected to perform classification of the lesions as melanoma or non-melanoma, according to the histological diagnosis. Having set the sensitivity in recognizing melanoma to 95%, ANN(1) resulted to be significantly better in the classification of reassuring lesions than ANN(2). This study suggests that multispectral image analysis and artificial neural networks could be used to support primary care physicians or general practitioners in identifying pigmented skin lesions that require further investigations.

Optical devices used for image analysis of pigmented skin lesions: a proposal for quality assurance protocol using tissue-like phantoms.

Different technological tools have been developed to aid in the diagnosis of pigmented skin lesions, including cameras working with conventional RGB colour systems, epiluminescence microscopy and spectrophotometric methods using visible and near infrared wavelengths. All the different procedures should provide in an objective and reproducible fashion quantitative measurements of the colour and shape features of a given skin mole. At present, many devices have been introduced in experimental stages for clinical diagnosis, mainly used to provide to the clinicians an objective, computer-assisted second opinion. As for any diagnostic instruments, optical devices should also be subjected to a dedicated quality assurance protocol in order to evaluate the response repeatability of each device (intra-instrument agreement) and to check the accordance among the responses of different devices (inter-instrument agreement). The aim of this study was to design a quality assurance protocol for optical devices dedicated to image analysis of pigmented skin lesions and, in case, to detect cutaneous melanoma by using suitable tissue-like phantoms as standard references that enable testing of both hardware and software components. As an example, we report the results of intra-instrument and inter-instrument agreement when the protocol was applied on a series of 30 SpectroShade instruments, a novel optical device based on multi-spectral image analysis of colour and shape features of pigmented skin lesion.

Photodynamic therapy by topical meso-tetraphenylporphinesulfonate tetrasodium salt administration in superficial basal cell carcinomas.

The efficacy of an originally developed photodynamic approach, using topical administration of tetraphenylporphinesulfonate as the photosensitizer, was evaluated in a series of 292 basal cell carcinoma lesions (less than 2-mm thick) in 50 treated patients. The lack of indication for conventional therapies was the main selection criterion. The photosensitizing agent (2% solution) was topically applied at 0.1 ml/cm2, followed by light irradiation with a dye laser emitting at 645 nm (120 or 150 J/cm2). After initial treatment, all lesions responded, with 273 (93.5%) complete responses. Recurrences were observed in 29 (10.6%). A second application of photoradiation was performed in 15 persistent lesions and 11 relapsed lesions, producing 19/26 complete responses. Our results suggest that this technique can be considered a promising alternative treatment modality in selected cases of superficial basal cell carcinomas.

The invisible colours of melanoma. A telespectrophotometric diagnostic approach on pigmented skin lesions.

Reflectance images of 43 pigmented lesions of the skin (18 melanomas, 17 common melanocytic naevi and eight dysplastic naevi) were acquired by a telespectrophotometric system and were analysed in the spectral range from 420 to 1040 nm, to discriminate melanoma from benign melanocytic entities. Different evaluations were carried out considering the whole spectrum, the visible and the near infra-red. A total of 33 (76.7%) lesions were correctly diagnosed by the telespectrophotometric system, compared with 35 (81.4%) correct clinical diagnoses. Reflectance in the infra-red band appears diagnostically relevant. A larger study is needed to prove the validity of this diagnostic method.

Sequential half-body irradiation as systemic treatment of progressive Ewing sarcoma.

Sequential half-body irradiation (HBI) to be delivered in two sessions was used in 18 consecutive patients with metastatic Ewing sarcoma who relapsed after radiotherapy and multidrug chemotherapy. The HBI program to both upper and lower hemi body was completed in 11 patients (61%). The remaining 7 patients received only one single treatment of HBI because of relapse before the completion of the treatment program. In 20 of the 29 sessions HBI was employed to treat overt metastases. The overall objective response rate was 50%. Six of 18 patients (33%) are alive from 4 to 27 months, 3 of them without evidence of disease. No severe toxicity was observed. HBI as systemic treatment was more effective in patients who relapsed while off chemotherapy, with metastases confined to the lungs or to one single bone segment.

Accuracy evaluation of fusion of CT, MR, and spect images using commercially available software packages (SRS PLATO and IFS).

PURPOSE: A problem for clinicians is to mentally integrate information from multiple diagnostic sources, such as computed tomography (CT), magnetic resonance (MR), and single photon emission computed tomography (SPECT), whose images give anatomic and metabolic information. METHODS AND MATERIALS: To combine this different imaging procedure information, and to overlay correspondent slices, we used commercially available software packages (SRS PLATO and IFS). The algorithms utilize a fiducial-based coordinate system (or frame) with 3 N-shaped markers, which allows coordinate transformation of a clinical examination data set (9 spots for each transaxial section) to a stereotactic coordinate system. The N-shaped markers were filled with fluids visible in each modality (gadolinium for MR, calcium chloride for CT, and 99mTc for SPECT). The frame is relocatable, in the different acquisition modalities, by means of a head holder to which a face mask is fixed so as to immobilize the patient. Position errors due to the algorithms were obtained by evaluating the stereotactic coordinates of five sources detectable in each modality. RESULTS: SPECT and MR position errors due to the algorithms were evaluated with respect to CT: deltax was < or = 0.9 mm for MR and < or = 1.4 mm for SPECT, deltay was < or = 1 mm and < or = 3 mm for MR and SPECT, respectively. Maximal differences in distance between estimated and actual fiducial centers (geometric mismatch) were in the order of the pixel size (0.8 mm for CT, 1.4 mm for MR, and 1.8 mm for SPECT). In an attempt to distinguish necrosis from residual disease, the image fusion protocol was studied in 35 primary or metastatic brain tumor patients. CONCLUSIONS: The image fusion technique has a good degree of accuracy as well as the potential to improve the specificity of tissue identification and the precision of the subsequent treatment planning.

A comparison of efficacy of photoradiation therapy and other conventional treatment modalities on experimental MS-2 sarcoma.

The therapeutic efficacy of photoradiation therapy (PRT) following hematoporphyrin derivative (HpD) administration was compared in the experimental MS-2 tumour model to that of conventional treatment methods for local control of neoplastic diseases. The therapeutic effects of PRT and surgical removal of primary tumour were comparable in these experiments. However, optimal effects were critically dependent on the stage of tumour development. In addition, the therapeutic advantage of PRT over radiotherapy suggest an interesting role of a new approach in tumours resistant to this conventional treatment.

Biodistribution of haematoporphyrin analogues in a lung carcinoma model.

In an attempt to identify novel compounds useful for the optimization of Photodynamic Therapy (PDT), the tissue localization of new synthetic porphyrins was compared with Photofrin II in nude mice xenografted with a human small cell lung cancer (POVD). Three haematoporphyrin analogues were selected for this study based on prior in vitro photosensitivity screening of a series of 15 such derivatives, as well as on the basis of improved localization in C6 gliomas in mice. Two of the porphyrins yielded better tumour:normal lung ratios than Photofrin II and, of these two, one (P13) is known to exhibit good photosensitization properties both in vitro and in vivo, and is therefore a good candidate as a lead compound for the development of porphyrins suitable for the photodynamic treatment of lung tumours.

Increasing incidence of cutaneous melanoma, ultraviolet radiation and the clinician.

Incidence of melanoma is certainly rising all over the world and this observation has been related to a more frequent and prolonged exposure to the rays of the sun. The authors critically review pertinent literature and conclude that descriptive epidemiology of melanoma does not give survival trends and does not support the claim that melanoma is ultraviolet (UV) dependent. Analytical epidemiology has not reached a consensus on this aspect. Experimental data available are also difficult to interpret because there are consistent differences of susceptibility to UV among different animals, among lamps used and methods of measuring employed in various laboratories. Information available shows that the maximal relative biological activity of UV in humans is at about 305 nm. This evaluation greatly depends on (1) thickness of the skin, (2) the quantity and quality of secretions that cover the skin, (3) cleanness of the skin, (4) the latitude, (5) the weather, (6) the hour of the day and (7) the presence of chemical carcinogens in the air and on the skin. The authors stress the importance of the criteria of clinical diagnosis recently introduced in clinical practice and higher public awareness as the causes of the increasing incidence of melanoma.

In vivo spectrophotometric evaluation of neoplastic and non-neoplastic skin pigmented lesions--I. Reflectance measurements.

Reflectance spectrophotometry from 400 to 800 nm on different cutaneous pigmented lesions, including primary and metastatic malignant melanoma, pigmented nevi, lentigo and seborrhoeic keratosis, has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of the different lesions manifest dissimilar patterns, particularly in the near IR region. Comparison of reflectance of nevi with that of malignant melanomas results in a highly significant difference (P less than 10(-6)) between the two samples. Though interpretation of the spectra remains difficult as a result of the complexity of the optical processes of scattering and absorption, our results suggest that a detailed analysis of the reflectance spectrum may give clinically useful information, and could be utilized as an aid in clinical diagnosis of cutaneous pigmented lesions, especially where malignant melanoma is concerned.

In vivo spectrophotometric evaluation of neoplastic and non-neoplastic skin pigmented lesions. III. CCD camera-based reflectance imaging.

Reflectance spectroscopy, which allows an objective evaluation of the color of surfaces, has recently been proposed as a useful tool to discriminate cutaneous melanoma from other pigmented cutaneous lesions. A novel spectrophotometric system based on the use of a charge coupled device camera provided with a set of interference filters has been developed to acquire images of cutaneous pigmented lesions at selected wavelengths ranging from 420 to 1040 nm. For each filter, an image was captured, digitized by a frame grabber and stored in a personal computer to perform off-line data handling. Reflectance images were acquired of 22 cutaneous pigmented lesions including melanoma and dysplastic, compound and junctional nevus. From each spectral image, three parameters, i.e. mean reflectance, variegation index and lesion area; were derived at the corresponding wavelength. The wavelength dependence of the three parameters was significantly different when melanoma was compared to the other investigated lesions. Although preliminary, our results suggest that telespectrophotometry gives useful information and could be utilized as an aid in the clinical diagnosis of cutaneous pigmented lesions.

In vivo spectrophotometric evaluation of neoplastic and non-neoplastic skin pigmented lesions. II: Discriminant analysis between nevus and melanoma.

Reflectance spectrophotometry from 420 to 780 nm on 31 primary melanoma and 31 benign nevi has been performed by using an external integrating sphere coupled to a spectrophotometer. Measurements show that reflectance spectra of melanoma and nevi manifest dissimilar patterns. From these spectra four variables, whose physical and/or physiological meanings remain to be investigated, have been derived. All of them are significantly different when compared between melanoma and nevi. A discriminant function between the two groups of lesions has been determined by using a stepwise discriminant analysis, resulting in a test with a sensitivity of 90.3% and a specificity of 77.4%. This method of discrimination between melanoma and nevi seems to have a discriminating power almost equal to that of a clinical judgement from a specialized medical doctor, thus suggesting a new method for screening skin pigmented lesions.

Optical imaging and automated melanoma detection: questions and answers.

Early detection and prompt excision of cutaneous melanoma is of paramount importance to improve patient survival, and the clinician should be aware of the clinical features that suggest the presence of a malignant lesion. The clinical diagnosis is mainly based on observation of the colour and shape of a given skin lesion. Unfortunately, evaluation of a pigmented lesion is to a large extent subjective and is closely related to the experience of the clinician. To overcome this problem, optical imaging techniques using different instrumentation (i.e. colour video camera, epiluminescence microscopy, reflectance spectrophotometry) and computer image analysis have been proposed in an attempt to provide quantitative measurements in an objective and reproducible fashion. The different procedures employed to perform the diagnosis automatically all have a common denominator: mimicking the eye and the brain of the clinician by image processing and computerized analysis programs, respectively. Sensitivity and specificity data reported in the literature suggest that the computer-based diagnosis of melanoma does not greatly differ from the diagnostic capability of an expert clinician, and is independent of the optical acquisition method employed to analyse the lesions. Most of the computer-processed morphometric variables useful in automated diagnosis are not recognizable nor can be objectively evaluated by the human eye, except that of lesion dimension. However, several questions should be answered before assessing the actual usefulness, including the potential and limitations, of computer-based diagnostic procedures. The purpose of this study was to briefly review the different kinds of instrumentation being used to diagnose melanoma, and to raise questions and whenever possible provide answers in an attempt to establish whether there will be a future for these computerized systems.

Radiobiological studies on the 65 MeV therapeutic proton beam at Nice using human tumour cells.

PURPOSE: To determine the relative biological effectiveness (RBE) for initial and delayed inactivation of cells by a modulated proton beam suitable for the treatment of tumours of the eye, within the spread-out Bragg peak and in its distal declining edge. MATERIALS AND METHODS: Human tumour SCC25 cells were irradiated with the 65 MeV proton beam at the Cyclotron Medicyc in Nice. Perspex plates of different thickness were used to simulate five positions along the beam line: 2mm corresponding to the entrance beam; 15.6 and 25 mm in the spread-out Bragg peak; 27.2 and 27.8mm for the distal edge. At each position clonogenic survival of the irradiated cells and of their progeny were determined at various dose values. 60Co gamma-rays were used as reference radiation. RESULTS: RBE values evaluated at the survival level given by 2 Gy of gamma-rays increased with increasing depth from close to 1.0 at the proximal to about 1.2 at the distal part of the peak. Within the declining edge it reached the value of about 1.4 at 27.2 and about 2 at 27.8 mm. For the progeny of irradiated cells, the RBE value ranged from 1.0 to 1.1 within the spread-out Bragg peak and then increased up to a value of 2.0 at the last position. The dose-effect curves for the progeny always had a larger shoulder than for the irradiated progenitors, their alpha parameters being lower by a factor of about 4 and their beta parameters always being higher. The alpha/beta ratio was about 50 Gy for the progenitors and about 6 Gy for their progeny. The incidence of delayed effects increased with dose and with the depth within the beam. CONCLUSIONS: RBE values for the inactivation of cells irradiated in the spread-out Bragg peak are compatible with the value currently assumed in clinical applications. In the distal declining edge of the beam, the RBE values increased significantly to an extent that may be of concern when the region of the treatment volume is close to sensitive tissues. The yield of delayed reproductive cell death was significant at each position along the beam line.

Inactivation of C3H10T1/2 cells by low energy protons and deuterons.

PURPOSE: To determine the RBE-LET relationship for C3H10T1/2 cell inactivation by protons in the LET range 11-33 keV/microm and to compare inactivation frequencies induced in C3H10T1/2 cells by protons and deuterons at two matching LET values in the range 11-20 keV/microm. MATERIALS AND METHODS: C3H10T1/2 cells were irradiated with protons and deuterons at the radiobiological facility set up at the 7MV Van de Graaff accelerator at the LNL, Legnaro, Padova. Gamma rays from 60Co were used as reference radiation. RESULTS: Proton RBE values (alpha/alphagamma) for inactivation of C3H10T1/2 cells are constant around a value of 2 between 11 and 20 keV/microm and then rise sharply to reach a value of 4.2+/-1.0 at 33 keV/microm. Deuteron RBE values are 1.7+/-0.4 and 2.2+/-0.6 at LET values of 13 and 18 keV/microm respectively. CONCLUSIONS: Proton RBE values with C3H10T1/2 cells are significantly larger than unity at LET values as low as 11 keV/microm. No difference in effectiveness for inactivation of C3H10T1/2 has been found between protons and deuterons at two LET values in the range 10-20 keV/microm.

Inactivation of human normal and tumour cells irradiated with low energy protons.

PURPOSE: To analyse the cell inactivation frequencies induced by low energy protons in human cells with different sensitivity to photon radiation. MATERIALS AND METHODS: Four human cell lines with various sensitivities to photon irradiation were used: the SCC25 and SQ20B derived from human epithelium tumours of the tongue and larynx, respectively, and the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The cells were irradiated with y-rays and proton beams with linear energy transfer (LET) from 7 to 33 keV/microm. Clonogenic survival was assessed. RESULTS: Survival curves are reported for each cell line following irradiation with gamma-rays and with various proton LETs. The surviving fraction after 2 Gy of gamma-rays was 0.72 for SQ20B cells, and 0.28-0.35 for the other cell lines. The maximum LET proton effectiveness was generally greater than that of gamma-rays. In particular there was a marked increase in beam effectiveness with increasing LET for the most resistant cells (SQ20B) whose 2 Gy-survival varied from 0.72 with gamma-radiation down to 0.37 with 30 keV/microm protons. The relative biological effectiveness (RBE(2 Gy gamma)) with the 30 keV/microm beam, evaluated as the ratio of 2 Gy to the proton dose producing the same inactivation level as that given by 2 Gy of gamma-rays, was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively. CONCLUSIONS: RBE for inactivation with high-LET protons increased with the cellular radioresistance to gamma-rays. The cell line with the greatest resistance to gamma-rays was the most responsive to the highest LET proton beam. A similar trend has also been found in studies reported in the literature with He, C, N ions with LET in the range 20-125 keV/microm on human tumour cell lines.

Monte Carlo simulation of light fluence in tissue in a cylindrical diffusing fibre geometry.

The propagation of light emitted by a linear light diffuser in a cylindrical hollow organ was investigated by means of the Monte Carlo (MC) method. The height and radius of the cavity, scattering (mu(s)) (or reduced scattering, mu'(s)) and absorption (mu(a)) coefficients, anisotropy (g), and refractive indices of the media involved (n1, n2) are required as input data by the MC code, as are characteristics of the light diffuser (length, delivered power and emission profile). Results of our MC model were tested by measuring the light fluence rate in a tissue-simulating phantom (mu(a) = 0.5 cm(-1), mu(s) = 23 cm(-1) and g = 0.75) irradiated at 633 nm with a cylindrical diffuser. Since geometric and optical parameters determine the behaviour of light propagation in tissue, MC simulations with different sets of input parameters were carried out to provide qualitative as well as quantitative data useful in planning photodynamic therapy. Data are reported on light penetration and fluence rate build-up at mu(a) and mu'(s) values ranging between 0.1 and 5 cm(-1) and 2.5 and 50 cm(-1), respectively. Furthermore, results suggest that a shift and spread could occur in the isofluence curves along the symmetry axis, which depend on the diameter of the treated lumen as well as on the emission profile of the light diffuser. Using our data it is possible to estimate how inaccuracy in knowledge of the optical coefficients can affect (i.e. usually by increasing) the light dose scheduled at a certain depth into tissue.