RESUMO
Pulsed dye lasers are used effectively in the treatment of psoriasis with long remission time and limited side effects. It is, however, not completely understood which biological processes underlie its favorable outcome. Pulsed dye laser treatment at 585-595 nm targets hemoglobin in the blood, inducing local hyperthermia in surrounding blood vessels and adjacent tissues. While the impact of destructive temperatures on blood vessels has been well studied, the effects of lower temperatures on the function of several cell types within the blood vessel wall and its periphery are not known. The aim of our study is to assess the functionality of isolated blood vessels after exposure to moderate hyperthermia (45 to 60°C) by evaluating the function of endothelial cells, smooth muscle cells, and vascular nerves. We measured blood vessel functionality of rat mesenteric arteries (n=19) by measuring vascular contraction and relaxation before and after heating vessels in a wire myograph. To this end, we elicited vascular contraction by addition of either high potassium solution or the thromboxane analogue U46619 to stimulate smooth muscle cells, and electrical field stimulation (EFS) to stimulate nerves. For measurement of endothelium-dependent relaxation, we used methacholine. Each vessel was exposed to one temperature in the range of 45-60°C for 30 seconds and a relative change in functional response after hyperthermia was determined by comparison with the response per stimulus before heating. Non-linear regression was used to fit our dataset to obtain the temperature needed to reduce blood vessel function by 50% (Half maximal effective temperature, ET50). Our findings demonstrate a substantial decrease in relative functional response for all three cell types following exposure to 55°C-60°C. There was no significant difference between the ET50 values of the different cell types, which was between 55.9°C and 56.9°C (P>0.05). Our data show that blood vessel functionality decreases significantly when exposed to temperatures between 55°C-60°C for 30 seconds. The results show functionality of endothelial cells, smooth muscle cells, and vascular nerves is similarly impaired. These results help to understand the biological effects of hyperthermia and may aid in tailoring laser and light strategies for selective photothermolysis that contribute to disease modification of psoriasis after pulsed dye laser treatment.
Assuntos
Lasers de Corante , Animais , Ratos , Masculino , Lasers de Corante/uso terapêutico , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/efeitos da radiação , Vasodilatação/efeitos da radiação , Vasodilatação/fisiologia , Temperatura , Músculo Liso Vascular/efeitos da radiação , Músculo Liso Vascular/fisiologia , Células Endoteliais/efeitos da radiação , Células Endoteliais/fisiologia , Vasoconstrição/efeitos da radiação , Vasoconstrição/fisiologia , Endotélio Vascular/efeitos da radiação , Ratos WistarRESUMO
Photogrammetry is a method for obtaining virtual 3D models of objects and scenes. The technique is increasingly used to record the crime scene in its original, undisturbed state for mapping, analytical and reconstruction purposes. Recently, it was shown that it is possible to visualize and/or chemically analyze latent traces by using advanced cameras which either operate in wavelength ranges beyond the visible range, and/or are able to obtain spectrally resolved images. The combination of these advanced cameras and photogrammetric techniques enables the 3D registration of valuable information. We successfully explored the feasibility to obtain visible, infrared, hyperspectral and thermal 3D registrations of simulated crime scenes using photogrammetry, and demonstrate the possibilities and practical challenges for use in forensic practice.
Assuntos
Ciências Forenses/métodos , Raios Infravermelhos , Fotogrametria , Análise Espectral , Termografia , Temperatura Corporal , Humanos , Imageamento TridimensionalRESUMO
Endoscopic surveillance is recommended for patients with Barrett's esophagus (BE) to detect high-grade intraepithelial neoplasia (HGIN) or early cancer (EC). Early neoplasia is difficult to detect with white light endoscopy and random biopsies are associated with sampling error. Fluorescence spectroscopy has been studied to distinguish non-dysplastic Barrett's epithelium (NDBE) from early neoplasia. The Optical Biopsy System (OBS) uses an optical fiber integrated in a regular biopsy forceps. This allows real-time spectroscopy and ensures spot-on correlation between the spectral signature and corresponding physical biopsy. The OBS may provide an easy-to-use endoscopic tool during BE surveillance. We aimed to develop a tissue-differentiating algorithm and correlate the discriminating properties of the OBS with the constructed algorithm to the endoscopist's assessment of the Barrett's esophagus. In BE patients undergoing endoscopy, areas suspicious for neoplasia and endoscopically non-suspicious areas were investigated with the OBS, followed by a correlating physical biopsy with the optical biopsy forceps. Spectra were correlated to histology and an algorithm was constructed to discriminate between HGIN/EC and NDBE using smoothed linear dicriminant analysis. The constructed classifier was internally cross-validated and correlated to the endoscopist's assessment of the BE segment. A total of 47 patients were included (39 males, age 66 years): 35 BE patients were referred with early neoplasia and 12 patients with NDBE. A total of 245 areas were investigated with following histology: 43 HGIN/EC, 66 low-grade intraepithelial neoplasia, 108 NDBE, 28 gastric or squamous mucosa. Areas with low-grade intraepithelial neoplasia and gastric/squamous mucosa were excluded. The area under the receiver operating characteristic curve of the constructed classifier was 0.78. Sensitivity and specificity for the discrimination between NDBE and HGIN/EC of OBS alone were 81% and 58% respectively. When OBS was combined with the endoscopist's assesssment, sensitivity was 91% and specificity 50%. If this protocol would have guided the decision to obtain biopsies, half of the biopsies would have been avoided, yet 4/43 areas containing HGIN/EC (9%) would have been inadvertently classified as unsuspicious. In this study, the OBS was used to construct an algorithm to discriminate neoplastic from non-neoplastic BE. Moreover, the feasibility of OBS with the constructed algorithm as an adjunctive tool to the endoscopist's assessment during endoscopic BE surveillance was demonstrated. These results should be validated in future studies. In addition, other probe-based spectroscopy techniques may be integrated in this optical biopsy forceps system.
Assuntos
Esôfago de Barrett/patologia , Biópsia/métodos , Detecção Precoce de Câncer/métodos , Neoplasias Esofágicas/patologia , Espectrometria de Fluorescência/métodos , Idoso , Algoritmos , Esôfago de Barrett/complicações , Carcinoma in Situ/etiologia , Carcinoma in Situ/patologia , Neoplasias Esofágicas/etiologia , Esofagoscopia/métodos , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e EspecificidadeRESUMO
In Barrett's esophagus (BE), second-generation autofluorescence imaging (AFI-II) improves targeted detection of high-grade intra-epithelial neoplasia (HGIN) and early cancer (EC), yet suffers from high false-positive (FP) rates. The newest generation AFI (AFI-III) specifically targets fluorescence in malignant cells and may therefore improve detection of early neoplasia and reduce FP rate. The aim was to compare AFI-III with AFI-II for endoscopic detection of early neoplasia in BE. BE patients with endoscopically inconspicuous neoplasia underwent two diagnostic endoscopies (AFI-II/AFI-III) in a single session. End-points: number of patients and lesions with HGIN/EC detected with AFI-II and AFI-III after white-light endoscopy (WLE) and the value of reinspection of AFI-positive areas with WLE and narrow-band imaging. Forty-five patients were included (38 males, age 65 years). Nineteen patients showed HGIN/EC. AFI-II inspection after WLE increased detection of HGIN/EC from 9 to 15 patients (47 to 79%); AFI-III increased detection from 9 to 17 patients (47 to 89%). WLE plus random biopsies diagnosed 13/19 (68%) HGIN/EC patients. One hundred and four abnormal AFI areas were inspected; 23 (22%) showed HGIN/EC. AFI-II increased detection of HGIN/EC from 10 to 18 lesions (43 to 78%). AFI-III increased detection from 10 to 20 lesions (43-87%). FP rate was 86% for AFI-II and AFI-III. Reinspection with WLE or narrow-band imaging reduced FP rate to 21% and 22%, respectively, but misclassified HGIN/EC lesions as unsuspicious in 54% and 31%, respectively. This first feasibility study on third-generation AFI again showed improved targeted detection of HGIN/EC in BE. However, the results do not suggest AFI-III performs significantly better than conventional AFI-II.
Assuntos
Esôfago de Barrett/patologia , Carcinoma in Situ/patologia , Neoplasias Esofágicas/patologia , Esofagoscopia/métodos , Imagem Óptica , Lesões Pré-Cancerosas/patologia , Idoso , Reações Falso-Positivas , Feminino , Humanos , Luz , Masculino , Imagem de Banda Estreita , Projetos PilotoRESUMO
Hyperspectral imaging (HSI) integrates conventional imaging and spectroscopy, to obtain both spatial and spectral information from a specimen. This technique enables investigators to analyze the chemical composition of traces and simultaneously visualize their spatial distribution. HSI offers significant potential for the detection, visualization, identification and age estimation of forensic traces. The rapid, non-destructive and non-contact features of HSI mark its suitability as an analytical tool for forensic science. This paper provides an overview of the principles, instrumentation and analytical techniques involved in hyperspectral imaging. We describe recent advances in HSI technology motivating forensic science applications, e.g. the development of portable and fast image acquisition systems. Reported forensic science applications are reviewed. Challenges are addressed, such as the analysis of traces on backgrounds encountered in casework, concluded by a summary of possible future applications.
Assuntos
Análise Espectral/métodos , Manchas de Sangue , Dermatoglifia , Medicina Legal/métodos , Cabelo/química , Humanos , Processamento de Imagem Assistida por Computador , Luminescência , Transtornos Relacionados ao Uso de Substâncias/diagnóstico , Propriedades de SuperfícieRESUMO
The autofluorescence of fingermarks is used for their detection. The components responsible for this autofluorescence are largely unknown. Thin layer chromatography and fluorescence spectroscopy were used to identify autofluorescent components and evaluate their forensic value. Based on our results, tryptophan is hypothesized to be a major contributor to the autofluorescence when part of peptides or proteins, id est, not in its free form. Part of the autofluorescence could be assigned to a kynurenine derivative. Pheophorbide A, a metabolite of chlorophyll, is inferred as a red fluorescent fingermark component. Chlorophyll is a plant pigment which implies that dietary information can potentially be retrieved from fingermarks.
Assuntos
Dermatoglifia , Fluorescência , Bilirrubina/química , Clorofila/análogos & derivados , Clorofila/química , Cromatografia em Camada Fina , Flavina-Adenina Dinucleotídeo/química , Humanos , Cinurenina/química , Luz , Fenilalanina/química , Feofitinas/química , Protoporfirinas/química , Riboflavina/química , Sebo/química , Espectrometria de Fluorescência , Suor/química , Tiamina/química , Triptofano/química , Tirosina/química , Raios Ultravioleta , Vitamina B 6/química , Xanturenatos/química , beta Caroteno/químicaRESUMO
Our aim was to show that the colour difference between brighter and darker red, occasionally observed as an oscillating boundary in the recipient and donor parts of an arterioarterial anastomosis in severe twin-twin transfusion syndrome (TTTS), is a consequence of natural differences in blood oxygenation and hematocrit developing between donor and recipient twins. As method we defined a theoretical model of the placenta with dimensions from pathology examination. From literature we determined the optical absorption and scattering properties of all tissue components, and hematocrit and oxygen saturation values for donor and recipient twins. From our placental model we simulated the spectrum of back-scattered light by standard Monte Carlo photon propagation computations and calculated the colour of chorionic arterial and venous blood vessels by applying the physics theory of colour perception. Our computations demonstrate that recipient arterial blood is somewhat brighter red than donor arterial blood. The strong colour differences seen after laser coagulation of all anastomoses but the arterioarterial were explained from an angiotensin II cut-off in the recipient due to obliteration of arteriovenous anastomoses, causing a temporary increase in recipient placental perfusion and hence in blood oxygenation. In conclusion, natural differences in recipient versus donor blood oxygen saturation and hematocrit in severe TTTS explain the observed colour differences between brighter and darker red observed in the recipient and donor parts of arterioarterial anastomoses.
Assuntos
Fístula Artério-Arterial/congênito , Transfusão Feto-Fetal/fisiopatologia , Oxigênio/sangue , Cor , Percepção de Cores , Feminino , Hematócrito , Humanos , Modelos Biológicos , Método de Monte Carlo , Placenta/irrigação sanguínea , GravidezRESUMO
The growing resistance against antifungal agents has renewed the search for alternative treatment modalities, and antimicrobial photodynamic inactivation (PDI) is a potential candidate. The cationic porphyrin 5-phenyl-10,15,20-Tris(N-methyl-4-pyridyl)porphyrin chloride (TriP[4]) is a photosensitizer that in combination with light can inactivate bacteria, fungi, and viruses. For future improvement of the efficacy of PDI of clinically relevant fungi such as Candida albicans, we sought to understand the working mechanism by following the response of C. albicans exposed to PDI using fluorescence confocal microscopy and freeze-fracture electron microscopy. The following events were observed under dark conditions: TriP[4] binds to the cell envelope of C. albicans, and none or very little TriP[4] enters the cell. Upon illumination the cell membrane is damaged and eventually becomes permeable for TriP[4]. After lethal membrane damage, a massive influx of TriP[4] into the cell occurs. Only the vacuole membrane is resistant to PDI-induced damage once TriP[4] passes the plasma membrane. Increasing the incubation time of C. albicans with TriP[4] prior to illumination did not increase the influx of TriP[4] into the cell or the efficacy of PDI. After the replacement of 100% phosphate-buffered saline (PBS) by 10% PBS as the medium, C. albicans became permeable for TriP[4] during dark incubation and the efficacy of PDI increased dramatically. In conclusion, C. albicans can be successfully inactivated by the cationic porphyrin TriP[4], and the cytoplasmic membrane is the target organelle. TriP[4] influx occurred only after cell death.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/efeitos da radiação , Fotoquimioterapia , Fármacos Fotossensibilizantes , Porfirinas/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Corantes Fluorescentes , Técnica de Fratura por Congelamento , Microscopia Confocal , Microscopia Eletrônica , Espectrometria de FluorescênciaRESUMO
Proteases of the plasminogen activator (PA) and matrix metalloproteinase (MMP) system play an important role in smooth muscle cell (SMC) migration and neointima formation after vascular injury. Inhibition of either PAs or MMPs has previously been shown to result in decreased neointima formation in vivo. To inhibit both protease systems simultaneously, a novel hybrid protein, TIMP-1.ATF, was constructed consisting of the tissue inhibitor of metalloproteinase-1 (TIMP-1) domain, as MMP inhibitor, linked to the receptor-binding amino terminal fragment (ATF) of urokinase. By binding to the u-PA receptor this protein will not only anchor the TIMP-1 moiety directly to the cell surface, it will also prevent the local activation of plasminogen by blocking the binding of urokinase-type plasminogen activator (u-PA) to its receptor. Adenoviral expression of TIMP-1.ATF was used to inhibit SMC migration and neointima formation in human saphenous vein segments in vitro. SMC migration was inhibited by 65% in Ad.TIMP-1.ATF-infected cells. Infection with adenoviral vectors encoding the individual domains, Ad.TIMP-1 and Ad.ATF, reduced migration by 32% and 52%, respectively. Neointima formation in saphenous vein organ cultures infected with Ad.TIMP-1.ATF was inhibited by 72% compared with 42% reduction after Ad.TIMP-1 infection and 34% after Ad.ATF infection. These data show that binding of TIMP-1.ATF hybrid protein to the u-PA receptor at the cell surface strongly enhances the inhibitory effect of TIMP-1 on neointima formation in human saphenous vein cultures.
Assuntos
Músculo Liso Vascular/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Inibidor Tecidual de Metaloproteinase-1/genética , Túnica Íntima/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Adenoviridae/genética , Animais , Células CHO/citologia , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Cricetinae , Meios de Cultivo Condicionados/farmacologia , Ativação Enzimática/efeitos dos fármacos , Citometria de Fluxo , Técnicas de Transferência de Genes , Humanos , Técnicas In Vitro , Metaloproteinase 13 da Matriz , Inibidores de Metaloproteinases de Matriz , Músculo Liso Vascular/citologia , Estrutura Terciária de Proteína/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Veia Safena/citologia , Veia Safena/efeitos dos fármacos , Veia Safena/metabolismo , Inibidor Tecidual de Metaloproteinase-1/farmacologia , Túnica Íntima/efeitos dos fármacosRESUMO
A phase zero evaluation of a new fluorescence imaging technique for diagnosing cervical intraepithelial neoplasia (CIN) was performed. The fluorescence imaging prototype performed quantitative imaging of Protoporphyrin induced by a topically applied aminolevulinic acid using double ratio (DR) fluorescence imaging technique developed by our group. A total of 38 patients were in the protocol, with 16 colposcopically selected for biopsy. Fluorescence images of these 16 patients were taken, 19 sites were biopsied, and the disease was staged histopathologically. DR fluorescence imaging of the cervix using our general purpose prototype appeared to be cumbersome but feasible. In four cases strongly localized fluorescent hotspots were observed at the location where the disease was colposcopically visible. In the other cases the fluorescence showed a more diffuse multifocal image. The value of the DR determined at the site of biopsy correlated in a statistically significant way with the histopathologically determined stage of the disease [Spearman rank correlation, r=0.881, p<0.001 (confidence interval 0.7044-0.9552)]. This suggests that noninvasive staging of CIN using this technique is feasible. We believe that the results of this study justify the development of a dedicated device that combines regular white light colposcopy with DR fluorescence imaging.
Assuntos
Colposcopia/métodos , Espectrometria de Fluorescência/métodos , Displasia do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/diagnóstico , Adulto , Ácido Aminolevulínico/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Método de Monte Carlo , Estadiamento de Neoplasias/métodos , Óptica e Fotônica , Protoporfirinas/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/patologiaRESUMO
The in vivo pharmacokinetics of protoporphyrin IX (PpIX) after administration of 5-aminolevulinic acid (ALA) cannot be described accurately by mathematical models using first-order rate processes. We have replaced first-order reaction rates by dose-dependent (Michaelis-Menten [MM]) reaction rates in a mathematical compartment model. Different combinations of first-order and dose-dependent reaction rates were evaluated to see which one would improve the goodness-of-fit to experimentally determined in vivo PpIX fluorescence kinetics as a function of concentration. The mathematical models that were evaluated are all based on a three-compartment model for drug distribution, conversion to PpIX and subsequent conversion to heme. Implementation of dose-dependent reaction rates improved the goodness-of-fit and enabled interpolation to other drug doses. For most data sets the time constant for delivery to the target cells turned out to be dose dependent. For all data sets the use of MM rates for the conversion of ALA to PpIX yielded better fits. The clearance of PpIX turned out to be a first-order process for all doses and types of administration. Fluorescence curves measured on a specific tissue type but obtained in different studies with different measurement techniques could be described with a single set of parameters.
Assuntos
Protoporfirinas/farmacocinética , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/farmacocinética , Animais , Relação Dose-Resposta a Droga , Fluorescência , Cinética , Matemática , Camundongos , Modelos Biológicos , Fotoquimioterapia , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/farmacocinética , Pele/efeitos dos fármacos , Pele/metabolismoRESUMO
Injury-induced neointimal development results from migration and proliferation of vascular smooth muscle cells (SMC). Cell migration requires controlled proteolytic degradation of extracellular matrix surrounding the cell. Plasmin is a major contributor to this process by degrading various matrix proteins directly, or indirectly by activating matrix metalloproteinases. This makes it an attractive target for inhibition by gene transfer. An adenoviral vector, Ad.ATF.BPTI, was constructed encoding a hybrid protein, which consists of the aminoterminal fragment (ATF) of urokinase-type plasminogen activator (u-PA) linked to bovine pancreas trypsin inhibitor (BPTI), a potent inhibitor of plasmin. This hybrid protein binds to the u-PA receptor, thereby inhibiting plasmin activity at the cell surface, and was found to be a potent inhibitor of cell migration in vitro. Local infection with Ad.ATF.BPTI of balloon-injured rat carotid artery resulted in detectable expression of ATF.BPTI mRNA and protein in the vessel wall. Morphometric analysis of arterial cross-sections revealed that delivery of Ad.ATF.BPTI to the carotid artery wall at the time of balloon injury inhibited neointima formation by 53% (P < 0.01) at 14 days and 19% (P = NS) at 28 days after injury when compared with control vector-infected arteries. Intima/media ratios were decreased by 60% (P < 0.01) and 35% (P < 0.05) at 14 and 28 days, respectively, when compared with control vector-infected arteries. Furthermore, a small but significant increase in medial area was found in the Ad.ATF.BPTI-treated arteries at 28 days (P < 0.05). These results show that local infection of the vessel wall with Ad.ATF.BPTI reduces neointima formation, presumably by inhibiting SMC migration, thereby offering a novel therapeutic approach to inhibiting neointima development.
Assuntos
Angioplastia com Balão/efeitos adversos , Aprotinina/genética , Estenose das Carótidas/prevenção & controle , Fibrinolisina/antagonistas & inibidores , Terapia Genética/métodos , Adenoviridae/genética , Animais , Aprotinina/metabolismo , Artérias Carótidas/metabolismo , Estenose das Carótidas/patologia , Estenose das Carótidas/terapia , Técnicas de Cultura de Células , Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos , Humanos , Masculino , Fragmentos de Peptídeos/genética , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Recidiva , Túnica Íntima/patologia , Túnica Média/patologia , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
In 75% of cases, ovarian carcinoma has already metastasized in the abdominal cavity at the time of diagnosis. For determination of the necessity for a supplementary therapy, in addition to surgical resection, it is important to localize and stage microscopical intraperitoneal metastases of the tumor. Intraperitoneal photodetection of tumor metastases is based on preferential tumor distribution of a fluorescent tumor marker. The time-dependent differences in drug concentration between tumor and normal (T/N) tissues can be used to visualize small tumors. We performed fluorescence measurements on abdominal organs and tumor in the peritoneal cavity of rats. 5-Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was used as the fluorescent marker. Three different drug doses (100, 25 and 5 mg/kg) were used and PpIX fluorescence profiles were followed up to 24 h after intravenous administration. Maximum T/N ratios were found 2-3 h after administration of ALA with all drug doses. A significant T/N tissue contrast was obtained for all abdominal organs tested after administration of 5 mg/kg.
Assuntos
Corantes Fluorescentes/farmacocinética , Mesalamina/farmacocinética , Neoplasias Peritoneais/metabolismo , Protoporfirinas/farmacocinética , Animais , Relação Dose-Resposta a Droga , Feminino , Transplante de Neoplasias , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/secundário , Ratos , Ratos Wistar , Espectrometria de Fluorescência , Distribuição TecidualRESUMO
Smooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.
Assuntos
Antifibrinolíticos/metabolismo , Técnicas de Transferência de Genes , Proteínas Luminescentes/genética , Inibidores da Tripsina/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Adenoviridae/genética , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Vírus da Encefalomiocardite/genética , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/metabolismo , Fibrinolíticos/metabolismo , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Pâncreas , Ativadores de Plasminogênio/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Veia Safena/citologia , Veia Safena/metabolismo , Inibidores da Tripsina/metabolismo , Inibidores da Tripsina/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
This study has investigated damage to the intraperitoneal organs of the rat after systemic (intraperitoneal and intravenous) administration of low doses of 5-aminolevulinic acid (ALA) and illumination with a standard white-light operating-room (o.r.) lamp. The study has been done within the framework of a larger study in which the possibility of using ALA for localization of small-volume macroscopically nonvisible peritoneal metastasis of ovarian tumors is being investigated. Fluorescence diagnostics are done in addition to the standard staging and localization procedures, either through a laparoscope or during laparotomy. In these circumstances, fluorescence diagnostics involve some risk of photosensitization of critical organs since a broad-band (o.r.) light source is used during the surgical procedures for illumination of the operating area. The drug dose and the time interval between administration of ALA and illumination are varied and normal tissues are examined both macroscopically and microscopically for damage. A relationship is demonstrated between the maximum tolerable dose (MTD) of ALA (defined as the dose that does not cause any tissue damage) and the time interval between administration and illumination. The white light that is used for illumination of the operating area is sufficient to induce damage to the peritoneal organs at relatively low ALA doses. The MDTs for 2, 6 and 16 h intervals are found to be respectively 1, 10 and 100 mg kg-1. The results are similar for both intraperitoneal and intravenous administration.
Assuntos
Ácido Aminolevulínico/farmacologia , Luz/efeitos adversos , Cavidade Peritoneal/efeitos da radiação , Animais , Feminino , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/patologia , Intestino Delgado/efeitos da radiação , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/efeitos da radiação , Dose Máxima Tolerável , Ratos , Ratos WistarRESUMO
A method is described for measuring optical properties and deriving chromophore concentrations from diffuse reflection measurements at the surface of a turbid medium. The method uses a diffusion approximation model for the diffuse reflectance, in combination with models for the absorption and scattering coefficients. An optical fibre-based set-up, capable of measuring nine spectra from 400 to 1050 nm simultaneously, is used to test the method experimentally. Results of the analyses of phantom and in vivo measurements are presented. These demonstrate that in the wavelength range from 600 to 900 nm, tissue scattering can be described as a simple power dependence of the wavelength and that the tissue absorption can be accurately described by the addition of water, oxy- and deoxyhaemoglobin absorption.
Assuntos
Análise Espectral/instrumentação , Análise Espectral/métodos , Humanos , Imagens de Fantasmas , Pele/metabolismoRESUMO
To study the long-term effects of dialysis fluids on the peritoneal cavity, an in vivo model for continuous peritoneal dialysis in rats was developed. Mini vascular access ports were implanted subcutaneously in the neck of the rats and an attached catheter was instilled into the peritoneal cavity. Rats were injected daily with 10 mL of standard 3.86% Dianeal or saline for a period up to 12 weeks. In the peritoneal cavity an initial increase in total cells was observed after 4 weeks of fluid instillation. This had declined after 12 weeks. A similar trend was also seen for macrophage and neutrophil numbers, whereas the percentage of lymphocytes kept increasing in time. An effect of fluid instillation was observed on the density and the morphology of the mesothelial monolayer of the rats. A higher density of cells was observed after 12 weeks, and foci of young mesothelial cells within activated mesothelium were found. The results show that the rat model presented can be compared with the situation in the peritoneal cavity of continuous ambulatory peritoneal dialysis (CAPD) patients, and therefore is suitable for intervention studies.