RESUMO
We have recently developed a model of pancreatic islet transplantation into a decellularized pancreatic tail in rats. As the pancreatic skeletons completely lack endothelial cells, we investigated the effect of co-transplantation of mesenchymal stem cells and endothelial cells to promote revascularization. Decellularized matrix of the pancreatic tail was prepared by perfusion with Triton X-100, sodium dodecyl sulfate and DNase solution. Isolated pancreatic islets were infused into the skeletons via the splenic vein either alone, together with adipose tissue-derived mesenchymal stem cells (adMSCs), or with a combination of adMSCs and rat endothelial cells (rat ECs). Repopulated skeletons were transplanted into the subcutaneous tissue and explanted 9 days later for histological examination. Possible immunomodulatory effects of rat adMSCs on the survival of highly immunogenic green protein-expressing human ECs were also tested after their transplantation beneath the renal capsule. The immunomodulatory effects of adMSCs were also tested in vitro using the Invitrogen Click-iT EdU system. In the presence of adMSCs, the proliferation of splenocytes as a response to phytohaemagglutinin A was reduced by 47% (the stimulation index decreased from 1.7 to 0.9, P = 0.008) and the reaction to human ECs was reduced by 58% (the stimulation index decreased from 1.6 to 0.7, P = 0.03). Histological examination of the explanted skeletons seeded only with the islets showed their partial disintegration and only a rare presence of CD31-positive cells. However, skeletons seeded with a combination of islets and adMSCs showed preserved islet morphology and rich vascularity. In contrast, the addition of syngeneic rat ECs resulted in islet-cell necrosis with only few endothelial cells present. Live green fluorescence-positive endothelial cells transplanted either alone or with adMSCs were not detected beneath the renal capsule. Though the adMSCs significantly reduced in vitro proliferation stimulated by either phytohaemagglutinin A or by xenogeneic human ECs, in vivo co-transplanted adMSCs did not suppress the post-transplant immune response to xenogeneic ECs. Even in the syngeneic model, ECs co-transplantation did not lead to sufficient vascularization in the transplant area. In contrast, islet co-transplantation together with adMSCs successfully promoted the revascularization of extracellular matrix in the subcutaneous tissue.
RESUMO
We previously developed a deep learning-based web service (IsletNet) for an automated counting of isolated pancreatic islets. The neural network training is limited by the absent consensus on the ground truth annotations. Here, we present a platform (IsletSwipe) for an exchange of graphical opinions among experts to facilitate the consensus formation. The platform consists of a web interface and a mobile application. In a small pilot study, we demonstrate the functionalities and the use case scenarios of the platform. Nine experts from three centers validated the drawing tools, tested precision and consistency of the expert contour drawing, and evaluated user experience. Eight experts from two centers proceeded to evaluate additional images to demonstrate the following two use case scenarios. The Validation scenario involves an automated selection of images and islets for the expert scrutiny. It is scalable (more experts, images, and islets may readily be added) and can be applied to independent validation of islet contours from various sources. The Inquiry scenario serves the ground truth generating expert in seeking assistance from peers to achieve consensus on challenging cases during the preparation for IsletNet training. This scenario is limited to a small number of manually selected images and islets. The experts gained an opportunity to influence IsletNet training and to compare other experts' opinions with their own. The ground truth-generating expert obtained feedback for future IsletNet training. IsletSwipe is a suitable tool for the consensus finding. Experts from additional centers are welcome to participate.
Assuntos
Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas , Prova Pericial , Projetos Piloto , Transplante das Ilhotas Pancreáticas/métodos , Redes Neurais de ComputaçãoRESUMO
Infusing pancreatic islets into the portal vein currently represents the preferred approach for islet transplantation, despite considerable loss of islet mass almost immediately after implantation. Therefore, approaches that obviate direct intravascular placement are urgently needed. A promising candidate for extrahepatic placement is the omentum. We aimed to develop an extracellular matrix skeleton from the native pancreas that could provide a microenvironment for islet survival in an omental flap. To that end, we compared different decellularization approaches, including perfusion through the pancreatic duct, gastric artery, portal vein, and a novel method through the splenic vein. Decellularized skeletons were compared for size, residual DNA content, protein composition, histology, electron microscopy, and MR imaging after repopulation with isolated islets. Compared to the other approaches, pancreatic perfusion via the splenic vein provided smaller extracellular matrix skeletons, which facilitated transplantation into the omentum, without compromising other requirements, such as the complete depletion of cellular components and the preservation of pancreatic extracellular proteins. Repeated MR imaging of iron-oxide-labeled pancreatic islets showed that islets maintained their position in vivo for 49 days. Advanced environmental scanning electron microscopy demonstrated that islets remained integrated with the pancreatic skeleton. This novel approach represents a proof-of-concept for long-term transplantation experiments.
RESUMO
Diabetic peripheral neuropathy (DPN) is a common complication of diabetes with potential severe consequences. Its pathogenesis involves hyperglycemia-linked mechanisms, which may include changes in the expression of neurotrophic growth factors. We analyzed the expression of 29 factors potentially related to nerve degeneration and regeneration in skin biopsies from 13 type 1 diabetic pancreas and kidney recipients with severe DPN including severe depletion of intraepidermal nerve fibers (IENF) in lower limb skin biopsies (group Tx1 1st examination). The investigation was repeated after a median 28-month period of normoglycemia achieved by pancreas transplantation (group Tx1 2nd examination). The same tests were performed in 13 stable normoglycemic pancreas and kidney recipients 6-12 years posttransplantation (group Tx2), in 12 matched healthy controls (group HC), and in 12 type 1 diabetic subjects without severe DPN (group DM). Compared to DM and HC groups, we found a significantly higher (p < 0.05-0.001) expression of NGF (nerve growth factor), NGFR (NGF receptor), NTRK1 (neurotrophic receptor tyrosine kinase 1), GDNF (glial cell-derived neurotrophic factor), GFRA1 (GDNF family receptor alpha 1), and GFAP (glial fibrillary acidic protein) in both transplant groups (Tx1 and Tx2). Enhanced expression of these factors was not normalized following the median 28-month period of normoglycemia (Tx1 2nd examination) and negatively correlated with IENF density and with electrophysiological indices of DPN (vibration perception threshold, electromyography, and autonomic tests). In contrast to our expectation, the expression of most of 29 selected factors related to neural regeneration was comparable in subjects with severe peripheral nerve fiber depletion and healthy controls and the expression of six factors was significantly upregulated. These findings may be important for better understanding the pathophysiology of nerve regeneration and for the development of intervention strategies.
Assuntos
Neuropatias Diabéticas/metabolismo , Transplante de Rim , Fatores de Crescimento Neural/metabolismo , Regeneração Nervosa/fisiologia , Transplante de Pâncreas , Pele/metabolismo , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Nervosas/metabolismo , Condução Nervosa/fisiologia , Pele/inervaçãoRESUMO
Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful (n = 61) and failed (n = 98) islet isolations. Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m2 (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.
Assuntos
Isquemia Fria/estatística & dados numéricos , Diabetes Mellitus Tipo 1/cirurgia , Edema/epidemiologia , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/cirurgia , Tempo de Internação/estatística & dados numéricos , Doadores de Tecidos/estatística & dados numéricos , Vasoconstritores/uso terapêutico , Índice de Massa Corporal , Hospitalização , Humanos , Imuno-Histoquímica , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas , Estudos RetrospectivosRESUMO
Three magnetic resonance (MR)/fluorescence imaging probes were tested for visualization, cellular distribution, and survival of labeled pancreatic islets in vitro and following transplantation. As T(1) contrast agents (CAs), gadolinium(III) complexes linked to ß-cyclodextrin (Gd-F-ßCD) or bound to titanium dioxide (TiO2 @RhdGd) were tested. As a T(2) CA, perovskite manganite nanoparticles (LSMO@siF@si) were examined. Fluorescein or rhodamine was incorporated as a fluorescent marker in all probes. Islets labeled with gadolinium(III) CAs were visible as hyperintense spots on MR in vitro, but detection in vivo was inconclusive. Islets labeled with LSMO@siF@si CA were clearly visible as hypointense spots or areas on MR scans in vitro as well as in vivo. All CAs were detected inside the islet cells by fluorescence. Although the vitality and function of the labeled islets was not impaired by any of the tested CAs, results indicate that LSMO@siF@si CA is a superior marker for islet labeling, as it provides better contrast enhancement within a shorter scan time.
Assuntos
Meios de Contraste/química , Corantes Fluorescentes/química , Gadolínio/química , Ilhotas Pancreáticas/metabolismo , Compostos de Manganês/química , Animais , Células Cultivadas , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Nanopartículas Metálicas/química , Microscopia de Fluorescência , Radiografia , Ratos , Ratos Endogâmicos Lew , Titânio/química , beta-Ciclodextrinas/químicaRESUMO
Labeling of pancreatic islets with superparamagnetic iron oxide (SPIO) nanoparticles enables their post-transplant monitoring by magnetic resonance imaging (MRI). Although the nanoparticles are incorporated into islet cells in culture, little is known about their fate in vivo. We studied the morphology of labeled islets after transplantation, aiming to identify the MRI contrast particles and their relationship to transplantation outcomes. Rat islets labeled with the ferucarbotran were transplanted into the liver or under the kidney capsule of syngeneic and allogeneic rats. After in vivo MRI, morphology was studied by light, fluorescence and transmission electron microscopy. Morphology of syngeneic islets transplanted beneath the kidney capsule vs into the liver was similar. Iron particles were almost completely eliminated from the endocrine cells and remained located in host-derived macrophages surrounding the vital islets for the entire study period. In the allogeneic model, islets lost their function and were completely rejected within nine days following transplantation in both transplant models. However, intercellular transport of the SPIO particles and subsequent MRI findings was different in the liver and kidney. In the liver, the decreasing number of islet-related MRI spots corresponded with clearance of iron particles in rejected islets; in contrast, with renal transplants extensive iron deposits with a high effect on MRI signal persisted in phagocytic cells beneath the capsule. We conclude that MRI detection of the iron contrast agent correlates with islet survival and function in islet transplantation into the liver, while it does not correlate in the case of transplantation beneath the renal capsule.
Assuntos
Meios de Contraste/farmacocinética , Dextranos/farmacocinética , Animais , Meios de Contraste/química , Dextranos/química , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Rim/metabolismo , Fígado/metabolismo , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Masculino , Ratos , Ratos Endogâmicos , Coloração e Rotulagem , Distribuição TecidualRESUMO
BACKGROUND: Organ pancreas transplantation represents the only method enabling long-term normalization of glucose metabolism in type-1 diabetic subjects so far. Unfortunately, surgical complications of this kind of therapy are still frequent. As a safer alternative, transplantation of isolated pancreatic islets was introduced at the Institute for Clinical and Experimental Medicine as a clinical experiment in the year 2005. METHODS AND RESULTS: We isolated the islets from pancreases of cadaveric donors which did not fulfil criteria to perform organ pancreas transplantation. Altogether, 36 islet implantations were performed in 28 C-peptide negative subjects suffering from type-1 diabetes by August 2010. In 15 subjects (21 implantations) the main indication was extremely instable course of diabetes due to the hypoglycaemia unawareness syndrome. In 5 and 3 cases, combined islet and kidney and islet and liver transplants were performed, respectively. In addition, islet autotransplantation was performed in 5 subjects undergoing total pancreatectomy. No patient died during the study period. In all but 1 patient with primary islet afunction, islet transplantation led to a complete cure of the hypoglycemia unawareness syndrome. Out of 15 patients, 11 subjects in this group showed a significant C-peptide production (> 0.2 pmol/ml) after 1 year. The mean insulin dose after allotransplantation decreased from 37 to 14 units per day and in 3 subjects, insulin therapy could be withdrawn. Serious technical complications occurred in 6 subjects, which only in 2 cases required surgical revision and did not cause long-term sequels. CONCLUSIONS: In comparison with organ pancreas transplantation, pancreatic islet transplantation represents a substantially safer method for restitution of endogenous insulin production. Though it eliminates serious hypoglycemic episodes in labile diabetes, complete insulin withdrawal is still often not possible. However, due to continuing progress in the laboratory techniques as well as in the transplant procedure itself, the results are steadily improving.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante das Ilhotas Pancreáticas , Adulto , Glicemia/análise , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Feminino , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Transplante das Ilhotas Pancreáticas/efeitos adversos , Transplante das Ilhotas Pancreáticas/instrumentação , Transplante das Ilhotas Pancreáticas/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: In vitro labeling of pancreatic islets by iron nanoparticles enables their detection as hypoitnense spots on serial magnetic resonance (MR) images. We report the first results of a pilot trial aiming to test the feasibility and safety of this technique in humans. METHODS: Islets were labeled in culture with 5 µL/mL ferucarbotran for 6 to 48 hr and transplanted into the portal vein (12 infusions) in 8 C-peptide negative recipients. The liver area was examined the next day and 1, 4, and 24 weeks posttransplant using a 3T MR scanner. RESULTS: In all recipients, significant C-peptide levels and near-normal HbA1c values were achieved with 50% to 80% insulin dose reduction. No side effects related to the labeling procedure were documented. Typically, a significant islet spot number decrease (on average 60%) was detected at week 1 with subsequent only slight decrease for up to 24 weeks. In two subjects with labeling period of less than 6 and 10 hr, only few islet spots were detected corresponding to poor islet visualization in phantoms labeled for the same period of time. CONCLUSION: Pancreatic islets (PI) visualization was safe and successful in all recipients but was less efficient if labeling period was less than 16 hr. Significant decrease of islet spots occurred at week 1, suggesting early islet destruction or impaired engraftment. Afterward, the islet spot numbers remained stable for up to 24 weeks. Data show that MR detection of ferucarbotran-labeled islets enables their long-term noninvasive visualization and correlates with sustained C-peptide production.
Assuntos
Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/patologia , Fígado/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Peptídeo C/sangue , Diabetes Mellitus/cirurgia , Hemoglobinas Glicadas/metabolismo , Humanos , Ilhotas Pancreáticas/anatomia & histologia , Imagens de Fantasmas , Veia Porta/anatomia & histologiaRESUMO
Increased ATP/ADP ratio resulting from enhanced glycolysis and oxidative phosphorylation represents a plausible mechanism controlling the glucose-stimulated insulin secretion (GSIS) in pancreatic beta-cells. Although specific bioenergetics might be involved, parallel studies of cell respiration and mitochondrial membrane potential (DeltaPsi(m)) during GSIS are lacking. Using high resolution respirometry and parallel DeltaPsi(m) monitoring by two distinct fluorescence probes we have quantified bioenergetics in rat insulinoma INS-1E cells representing a suitable model to study in vitro insulin secretion. Upon glucose addition to glucose-depleted cells we demonstrated a simultaneous increase in respiration and DeltaPsi(m) during GSIS and showed that the endogenous state 3/state 4 respiratory ratio hyperbolically increased with glucose, approaching the maximum oxidative phosphorylation rate at maximum GSIS. Attempting to assess the basis of the "toxic" effect of fatty acids on insulin secretion, GSIS was studied after linoleic acid addition, which diminished respiration increase, DeltaPsi(m) jump, and magnitude of insulin release, and reduced state 3/state 4 dependencies on glucose. Its effects were due to protonophoric function, i.e. uncoupling, since without glucose, linoleic acid accelerated both state 3 and state 4 respiration by similar extent. In turn, state 3 respiration increased marginally with linoleic acid at 10-20mM glucose. We conclude that upon glucose addition in physiological range, the INS-1E cells are able to regulate the oxidative phosphorylation rate from nearly zero to maximum and that the impairment of GSIS by linoleic acid is caused by mitochondrial uncoupling. These findings may be relevant to the pathogenesis of type 2 diabetes.
Assuntos
Glucose/farmacologia , Insulinoma/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Ácido Linoleico/farmacologia , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Células Tumorais CultivadasRESUMO
In vitro labeling of pancreatic islets with iron nanoparticles enables their direct posttransplant visualization by magnetic resonance; however, there is still a discrepancy in the fate of iron nanoparticles. This study was performed to detail the labeling process, consequently to improve the labeling efficacy and to confirm safety for islet cells. The islets were visible on T2*-weighted magnetic resonance images as hypointense spots immediately after 1-hr cultivation. Although at this time already the sufficient superparamagnetic effect was achieved, most of the particles were deposed in islet macrophages and only later were they found in endosomes of endocrine islet cells. The iron content depended on length of culture period. The labeled islets showed an intact ultrastructure, responded normally to glucose stimulation in vitro, and were able to treat experimental diabetes. For purpose of subsequent magnetic resonance imaging, a 24-hr culture with ferucarbotran leads to sufficient labeling with no apparent adverse effect on beta cell morphology or function.
Assuntos
Compostos Férricos , Ilhotas Pancreáticas/patologia , Nanopartículas Metálicas , Animais , Células Cultivadas , Células Secretoras de Insulina/patologia , Transplante das Ilhotas Pancreáticas/métodos , Macrófagos/patologia , Imageamento por Ressonância Magnética/métodos , Ratos , Coloração e Rotulagem/métodos , Fatores de TempoRESUMO
BACKGROUND: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis. OBJECTIVES: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens. SUBJECTS: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study. METHODS: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247-279, 509-528, and 524-543; proinsulin a.a. 9-23; and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853-872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-gamma, tumor necrosis factor beta, transforming growth factor beta1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray. RESULTS: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-gamma (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect. CONCLUSION: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.
Assuntos
Citocinas/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Análise Serial de Proteínas , Adolescente , Sequência de Aminoácidos , Autoanticorpos/genética , Criança , Pré-Escolar , Família , Feminino , Glutamato Descarboxilase/química , Glutamato Descarboxilase/genética , Humanos , Interleucinas/genética , Masculino , Dados de Sequência MolecularRESUMO
BACKGROUND: Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-gamma secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] >/= 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-gamma, tumour necrosis factor [TNF]-beta, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-beta], IL-10) and inflammatory associated cytokines (TNF-alpha, IL-1alpha,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-gamma [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD(65)) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique. RESULTS: The Th1 cytokines IFN-gamma and TNF-beta, secreted both spontaneously and by GAD(65)- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-alpha and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-beta and IL-10 were elevated in T1D children compared to high-risk children. CONCLUSION: High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D.
Assuntos
Citocinas/sangue , Diabetes Mellitus Tipo 1/sangue , Predisposição Genética para Doença , Mediadores da Inflamação/sangue , Linfócitos T Reguladores/metabolismo , Células Th1/metabolismo , Adolescente , Biomarcadores/sangue , Peptídeo C/sangue , Quimiocinas/sangue , Criança , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , MasculinoRESUMO
A site-specific glycosylation of salivary glands (SGs) isolated from unfed and partially fed Ixodes ricinus females was identified with the use of lectin affinity labeling on sections and western blots of SDS-PAGE gels. The results revealed that secretory granules of a, b, and c cells of the type II acinus and e and f cells of the type III acinus are glycosylated. In partially engorged tick SGs, 2 subtypes of c cells were distinguished. The granules of c1 cells contained mannose, N-acetyl-D-glucosamine, and sialic acid residues. The granules of b, c2, and e cells exhibited complex glycoconjugates rich in mannose, N-acetyl-D-glucosamine, galactose, N-acetyl-D-galactosamine, and a moderate amount of sialic acid. The granules of f cells contained N-acetyl-D-glucosamine and mannose moieties. Type III acini surfaces were covered with mannose-specific ConA binding sites. Except the granules of salivary cells, sialic acid-specific lectins MAA II and SNA strongly bound cuticular structures of alveolar ducts, and weakly with the cuticular spiral thread of excretory salivary ducts. The total sialic acid level in SG homogenates isolated from partially fed females was determined by the thiobarbituric acid method. Sialic acid, which has been found during the development of a few insect species, has not been reported in ticks as yet.
Assuntos
Carboidratos/análise , Ixodes/metabolismo , Ixodes/ultraestrutura , Lectinas/metabolismo , Animais , Feminino , Glicosilação , Microscopia Eletrônica , Microscopia de Fluorescência , Ácido N-Acetilneuramínico/análise , Glândulas Salivares/citologia , Glândulas Salivares/metabolismo , Glândulas Salivares/ultraestrutura , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
BACKGROUND: We have recently described a magnetic resonance (MR) method for detection of rat pancreatic islets transplanted into the liver after labeling with superparamagnetic iron oxide nanoparticles. The aim of this work was to study whether this technique could be applicable over a longer period after transplantation and whether it could help to detect islet rejection. METHODS: Islets from Lewis and Wistar rats were cultured in the presence of iron oxide nanoparticles. Two thousand of Lewis (n=6) or Wistar (n=8) iron-labeled islets were transplanted into the portal vein of Lewis diabetic animals. Serial MR imaging of the liver were performed at 1, 2, 3, 4, 5, and 6 weeks. RESULTS: Although all allogeneic islets were rejected by 12 days, syngeneic animals remained normoglycemic throughout the study. At week 1, the labeled islets were visualized on MR scans as distinct hypointense spots homogeneously distributed in the liver. While their number declined only insignificantly in the syngeneic group, in the allogeneic group the number of spots gradually decreased until approximately 35% of their initial count. Although syngeneic islets showed a normal histology, the allogeneic islets were completely rejected. Iron particles, localized in macrophages, were detected only in the syngeneic islets and were absent in the rejected islet structures. In vitro incubation tests did not reveal any differences in insulin secretion between labeled and nonlabeled islets. CONCLUSIONS: MR imaging of iron-labeled pancreatic islets can be used for verification of the technical success of the transplantation procedure itself and for the detection of the decreasing relative islet mass due to rejection.