Uptake of liposomes by Peyer´s patches following per os administration in mice

O objetivo deste estudo foi marcar uma proteína utilizada como modelo, a soro albumina bovina (SAB), com 99mTecnécio (99mTc), encapsular a rroteína marcada (99mTc-SAB) em lipossomas e empregar este marcador para quantificar a 99m c-SAB capturada pelas placas de Peyer de camundongos Balb/c após administração oral. A 99mTc-SAB (taxa de marcação=94,9±2,4%; n=25) foi encapsulada em lipossomas multilamelar (MLV), unilamelar de pequeno tamanho (SUV) ou unilamelar de grande tamanho (LUV) compostos de fosfatidilcolina de soja (PC); PC e colesterol (CH) (razão molar 8/2); PC, CH e fosfatidilglicerol (PG) (razão molar 7/2/1) ou distearoilfosfatidilcolina (DSPC), CH e PG (razão molar 7/2/1). Utilizando eletroforese em gel de agarose e imunoeletroforese contra uma imunoglobulina anti-SAB específica, foi verificado que a SAB não sofreu degradação após ter sido marcada com 99mTc e encapsulada em lipossomas. 99mTc-SAB encapsulada em SUV compostos por PC/CH/PG ou DSPC/CH/ PG foi preferencialmente capturada pelas placas de Peyer. A captura de 99mTc-SAB encapsulada em LUV foi reduzida quando comparada com aquela observada para SUV com a mesma composição em lipídios. 99mTc-SAB encapsulada em MLV compostos por PC/CH/PG ou DSPC/CH/PG e em SUV compostos de PC ou PC/CH não foi capturada pelas placas de Peyer. Os resultados indicam que SUV pode ser um carreador para antígenos instáveis no trato gastrintestinal, tornando-os potencialmente capazes de induzir a resposta imunológica das mucosas.The aim of this study was to employ a model protein, the bovine serum albumin (SAB), radiolabelled with 99mTechnetium (99mTc) (99mTc-SAB) and encapsulated into liposomes, in order to determine the amount de 99mTc-SAB taken up by Peyer´s patches following per os administration in mice Balb/c. The 99mTc-SAB, labelling yield of 94.9±2.4 % (n=25), was encapsulated within multilamellar large vesicles (MLV), small unilamellar vesicles (SUV) or large unilamellar vesicles (LUV) composed of soybean phosphatidylcholine (PC); PC and cholesterol (CH) (molar ratio 8/2); PC, CH and phosphatidylglicerol (PG) (molar ratio 7/2/1) or distearoylphosphatidylcholine (DSPC), CH and PG (molar ratio 7/2/1). A gel electrophoresis and a gel immunoelectrophoresis, using a specific anti-SAB serum immunoglobulin, were employed to evaluate the integrity of SAB after labelling with 99mTc and after encapsulation into liposomes. Labelling with 99mTc and liposome encapsulation did not result in degradation of SAB. SUVencapsulated-99mTc-SAB was preferentially taken up by Peyer´s patches. Uptake of LUV-encapsulated-99mTc-SAB by Peyer´s patches was reduced when compared with that observed for the SUV-encapsulated-99mTc-SAB. 99mTc-SAB encapsulated into SUV composed of PC or PC/CH was not uptaked by Peyer´s patches. The lipid composition (PC or DSPC/CH/PG) of SUV did not modify 99mTc-SAB uptake by Peyer´s patches. In conclusion, SUV might be an effective carrier for unstable antigens administered per os to induce mucosal immune response

Classificação de hidrópsia endolinfática em pacientes com suspeita de doença de Menière utilizando ressonância magnética

Mestrado em Radiações Aplicadas às Tecnologias da Saúde - Ramo de especialização: Imagem por Ressonância Magnética.A doença de Ménière (DM) é uma patologia idiopática do ouvido interno, caracterizada por episódios de vertigem recorrentes, hipoacusia flutuante e acufenos. Esta tríade de sintomas foi descrita pela primeira vez em 1861 por Prosper Ménière, apontando a disfunção do ouvido interno como a origem do complexo de sintomas auditivo e vestibular. Quando a etiologia de Hidrópsia Endolinfática (HE) é conhecida, designa-se por Síndrome de Ménière (SM). A classificação morfológica da HE no vestíbulo e na cóclea é feita com recurso à utilização de imagem por Ressonância Magnética (RM) através da escala de Nagoya, 2008. Esta dissertação tem como objetivos compreender os benefícios da utilização da RM na Doença de Ménière, avaliar a sensibilidade da RM para a diferenciação entre ouvido sintomático e ouvido assintomático e comparar a sintomatologia associada a estes pacientes com o grau atribuído pelo observador através da análise das imagens de RM. Para a elaboração deste estudo foi utilizada uma amostra de 51 pacientes que foram submetidos a exame de RM dos ouvidos. Para a aquisição das imagens de RM foi utilizado um protocolo específico com administração de contraste endovenoso (Gadolíneo) e 4h após a sua injeção foram incluídas as sequências específicas, sendo estas T1 3D IR e 3D Fluid Atennuation Inversion Recovery (3D-FLAIR). As regiões de interesse para a análise deste estudo foram o vestíbulo e a cóclea. A RM evidenciou uma sensibilidade de 74,5% para a deteção da DM em pacientes com sintomas característicos da patologia, bem como que um maior numero de sintomas associados à mesma é fator preponderante para um diagnóstico de DM confirmado pelas imagens de RM. A RM permite a realização de um diagnóstico diferencial para a caracterização de um paciente com ouvido sintomático.ABSTRACT - Ménière's disease (MD) is an idiopathic pathology of the inner ear, characterized by episodes of recurrent vertigo, fluctuating hearing loss, and tinnitus. This triad of symptoms was first described in 1861 by Prosper Ménière, pointing to dysfunction of the inner ear as the origin of the complex of auditory and vestibular symptoms. When the etiology of Endolymphatic Hydropsy (HE) is known, it is called Ménière's Syndrome (SM). The morphological classification of HE in the vestibule and cochlea is made using the Magnetic Resonance Imaging (MR) image using the Nagoya scale, 2008. This dissertation aims to understand the benefits of using Ménière's Disease, to evaluate MRI sensitivity for the differentiation between the symptomatic ear and asymptomatic ear, and to compare the symptomatology associated with these patients with the degree assigned by the observer through the analysis of MRI. For the preparation of this study, a sample of 51 patients who underwent MRI of the ears was used. For the acquisition of MR imaging, a specific protocol with intravenous contrast administration (Gadolíneo) and 4h after its injection were included in the specific sequences, being these T1 3D IR and 3D Fluid Attenuation Inversion Recovery (3D-FLAIR). The regions of interest for the analysis of this study were the vestibule and the cochlea. MRI showed a sensitivity of 74.5% for the detection of DM in patients with symptoms characteristic of the pathology, as well as a greater number of symptoms associated with it, which is a preponderant factor for a diagnosis of DM confirmed by MR images. MRI allows the performance of a differential diagnosis for the characterization of a patient with the symptomatic ear.N/

Hank’s balanced salt solution: an alternative resuspension medium to label autologous leukocytes. Experience in inflammatory bowel disease

In this work Hank's balanced salt solution (HBSS) has been used, as resuspension medium, instead of leukocyte poor plasma (LPP) to label autologous white blood cells in 28 patients with suspicion af active inflammatory bowel disease.Labelled cells were reinjected and anterior and caudo-craneal views were obtained at 30 min, 2 h and 6 h p.i. Regions of interest were outlined on liver, spleen, lung, bone marrow (spine), background and lesions and the organ/background activity ratios were calculated in all scans. Patients were classified into 2 groups: Group 1: LPP, 30 patients and Groups 2: HBSS, 28 patients. Labelling efficiency was higher in HBBS group (89.0 ± 3.2 %) than in the LPP group (6.5 ± 6.3%). Organ/background activity ratios were similar in both groups. Concerning diagnostic accuracy was similar at 30 min and 2 h but the false positive rate increased at 6 h p.i. in the HBSS group. HBSS seems to be a valid alternative as resuspension medium in the labeling of autologous leukocytes but leukocyte poor plasma seem to induce less leukocyte damage. Based on these results, in our center HBSS is the currently used medium to label leukocytes

Hank?s balanced salt solution: an alternative resuspension medium to label autologous leukocytes. Experience in inflammatory bowel disease

In this work Hank's balanced salt solution (HBSS) has been used, as resuspension medium, instead of leukocyte poor plasma (LPP) to label autologous white blood cells in 28 patients with suspicion af active inflammatory bowel disease.Labelled cells were reinjected and anterior and caudo-craneal views were obtained at 30 min, 2 h and 6 h p.i. Regions of interest were outlined on liver, spleen, lung, bone marrow (spine), background and lesions and the organ/background activity ratios were calculated in all scans. Patients were classified into 2 groups: Group 1: LPP, 30 patients and Groups 2: HBSS, 28 patients. Labelling efficiency was higher in HBBS group (89.0 ± 3.2 %) than in the LPP group (6.5 ± 6.3%). Organ/background activity ratios were similar in both groups. Concerning diagnostic accuracy was similar at 30 min and 2 h but the false positive rate increased at 6 h p.i. in the HBSS group. HBSS seems to be a valid alternative as resuspension medium in the labeling of autologous leukocytes but leukocyte poor plasma seem to induce less leukocyte damage. Based on these results, in our center HBSS is the currently used medium to label leukocytes