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.
BACKGROUND: Transplantation of pancreatic islets into subcutaneous cavities in diabetic rats may be as or even more effective than transplantation into the portal vein. Identifying the optimal timing of the individual steps in this procedure is critical. METHODS: Macroporous scaffolds were placed in the subcutaneous tissue of diabetic male Lewis rats for 7 or 28 d and the healing of the tissue inside the scaffolds was monitored. A marginal syngeneic graft comprising 4 islets/g of recipient body weight was transplanted at the best timing focusing mainly on vascularization. Recipients were monitored for blood glucose levels and tolerance tests. Histological examination was performed in all implanted scaffolds. The presence of individual endocrine cells was analyzed in detail. RESULTS: Blood glucose levels remained within the physiological range in all recipients until the end of experiment as well as body weight increase. Coefficients of glucose assimilation were normal or slightly reduced with no statistically significant differences between the groups 40 and 80 d after transplantation. Histological analysis revealed round viable islets in the liver similar to those in pancreas, but alpha cells practically disappeared, whereas islets in the scaffolds formed clusters of cells surrounded by rich vascular network and the alpha cells remained partially preserved. CONCLUSIONS: Subcutaneous transplantation of pancreatic islets is considerably less invasive but comparably efficient as commonly used islet transplantation into the portal vein. In consideration of alpha and beta cell ratio, the artificial subcutaneous cavities represent a promising site for future islet transplantation therapy.
- MeSH
- experimentální diabetes mellitus * chirurgie MeSH
- krevní glukóza MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky * krevní zásobení chirurgie MeSH
- potkani inbrední LEW MeSH
- subkutánní tkáň MeSH
- transplantace Langerhansových ostrůvků * metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.
- MeSH
- beta-buňky metabolismus ultrastruktura MeSH
- chemická frakcionace MeSH
- cytoplazmatická granula metabolismus MeSH
- exprese genu * MeSH
- glukosa metabolismus MeSH
- inzulin genetika metabolismus MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- průtoková cytometrie metody MeSH
- zinek metabolismus MeSH
- zinkový transportér 8 MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Instant Blood-Mediated Inflammatory Reaction (IBMIR) is a major cause of graft loss during pancreatic islet transplantation, leading to a low efficiency of this treatment method and significantly limiting its broader clinical use. Within the procedure, transplanted islets obstruct intrahepatic portal vein branches and consequently restrict blood supply of downstream lying liver tissue, resulting typically in ischemic necrosis. The extent of ischemic lesions is influenced by mechanical obstruction and inflammation, as well as subsequent recanalization and regeneration capacity of recipient liver tissue. Monitoring of immediate liver perfusion impairment, which is directly related to the intensity of post-transplant inflammation and thrombosis (IBMIR), is essential for improving therapeutic and preventive strategies to improve overall islet graft survival. In this study, we present a new experimental model enabling direct quantification of liver perfusion impairment after pancreatic islet transplantation using ligation of hepatic arteries followed by contrast-enhanced magnetic resonance imaging (MRI). The ligation of hepatic arteries prevents the contrast agent from circumventing the portal vein obstruction and enables to discriminate between well-perfused and non-perfused liver tissue. Here we demonstrate that the extent of liver ischemia reliably reflects the number of transplanted islets. This model represents a useful tool for in vivo monitoring of biological effect of IBMIR-alleviating interventions as well as other experiments related to liver ischemia. This technical paper introduces a novel technique and its first application in experimental animals.
- MeSH
- embolie * komplikace diagnóza MeSH
- ischemie * diagnostické zobrazování etiologie MeSH
- játra * krevní zásobení diagnostické zobrazování patologie MeSH
- krysa rodu rattus MeSH
- magnetická rezonanční angiografie metody MeSH
- přežívání štěpu MeSH
- reprodukovatelnost výsledků MeSH
- teoretické modely MeSH
- transplantace Langerhansových ostrůvků škodlivé účinky MeSH
- vena portae * MeSH
- vylepšení obrazu metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Subcutaneously implanted polymeric scaffolds represent an alternative transplantation site for pancreatic islets (PIs) with the option of vascularisation enhancement by mesenchymal stem cells (MSC). Nevertheless, a proper timing of the transplantation steps is crucial. In this study, scaffolds supplemented with plastic rods were implanted into diabetic rats and two timing schemes for subsequent transplantation of bioluminescent PIs (4 or 7 days after rod removal) were examined by multimodal imaging. The cavities were left to heal spontaneously or with 10 million injected MSCs. Morphological and vascularisation changes were examined by MRI, while the localisation and viability of transplanted islets were monitored by bioluminescence imaging. The results show that PIs transplanted 4 days after rod removal showed the higher optical signal and vascularisation compared to transplantation after 7 days. MSCs slightly improved vascularisation of the graft but hindered therapeutic efficiency of PIs. Long-term glycaemia normalisation (4 months) was attained in 80% of animals. In summary, multimodal imaging confirmed the long-term survival and function of transplanted PIs in the devices. The best outcome was reached with PIs transplanted on day 4 after rod removal and therefore the suggested protocol holds a potential for further applications.
- MeSH
- alografty MeSH
- experimentální diabetes mellitus * diagnostické zobrazování chirurgie MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky * krevní zásobení diagnostické zobrazování metabolismus patologie MeSH
- luminiscenční měření * MeSH
- mezenchymální kmenové buňky metabolismus patologie MeSH
- potkani transgenní MeSH
- přežívání štěpu * MeSH
- tkáňové podpůrné struktury * MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- transplantace mezenchymálních kmenových buněk MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cell reprogramming requires efficient delivery of reprogramming transcription factors into the cell nucleus. Here, we compared the robustness and workload of two protein delivery methods that avoid the risk of genomic integration. The first method is based on fusion of the protein of interest to a protein transduction domain (PTD) for delivery across the membranes of target cells. The second method relies on de novo synthesis of the protein of interest inside the target cells utilizing synthetic mRNA (syn-mRNA) as a template. We established a Cre/lox reporter system in three different cell types derived from human (PANC-1, HEK293) and rat (BRIN-BD11) tissues and used Cre recombinase to model a protein of interest. The system allowed constitutive expression of red fluorescence protein (RFP), while green fluorescence protein (GFP) was expressed only after the genomic action of Cre recombinase. The efficiency of protein delivery into cell nuclei was quantified as the frequency of GFP+ cells in the total cell number. The PTD method showed good efficiency only in BRIN-BD11 cells (68%), whereas it failed in PANC-1 and HEK293 cells. By contrast, the syn-mRNA method was highly effective in all three cell types (29-71%). We conclude that using synthetic mRNA is a more robust and less labor-intensive approach than using the PTD-fusion alternative.
- MeSH
- buněčné jádro metabolismus MeSH
- buněčné klony MeSH
- DNA metabolismus MeSH
- genetické inženýrství MeSH
- HEK293 buňky MeSH
- integrasy metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- proteinové domény MeSH
- průtoková cytometrie MeSH
- rekombinantní fúzní proteiny chemie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- technika přenosu genů * MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: In vitro labelling of cells and small cell structures is a necessary step before in vivo monitoring of grafts. We modified and optimised a procedure for pancreatic islet labelling using bimodal positively charged poly(lactic-co-glycolic acid) nanoparticles with encapsulated perfluoro crown ethers and indocyanine green dye via microporation and compared the method with passive endocytosis. RESULTS: Pancreatic islets were microporated using two pulses at various voltages. We tested a standard procedure (poration in the presence of nanoparticles) and a modified protocol (pre-microporation in a buffer only, and subsequent islet incubation with nanoparticles on ice for 10 min). We compared islet labelling by microporation with labelling by endocytosis, i.e. pancreatic islets were incubated for 24 h in a medium with suspended nanoparticles. In order to verify the efficiency of the labelling procedures, we used (19)F magnetic resonance imaging, optical fluorescence imaging and confocal microscopy. The experiment confirmed that microporation, albeit fast and effective, is invasive and may cause substantial harm to islets. To achieve sufficient poration and to minimise the reduction of viability, the electric field should be set at 20 kV/m (two pulses, 20 ms each). Poration in the presence of nanoparticles was found to be unsuitable for the nanoparticles used. The water suspension of nanoparticles (which served as a surfactant) was slightly foamy and microbubbles in the suspension were responsible for sparks causing the destruction of islets during poration. However, pre-microporation (poration of islets in a buffer only) followed by 10-min incubation with nanoparticles was safer. CONCLUSIONS: For labelling of pancreatic islets using poly(lactic-co-glycolic acid) nanoparticles, the modified microporation procedure with low voltage was found to be safer than the standard microporation procedure. The modified procedure was fast, however, efficiency was lower compared to endocytosis.
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Pancreas transplantation (PTx) represents the method of choice in type 1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. In 2005, the Institute for Clinical and Experimental Medicine (IKEM) launched a program to investigate the safety potential of islet transplantation (ITx) in comparison to PTx. AIM: This study aims to compare the results of PTx and ITx regarding severe hypoglycemia elimination, metabolic control, and complication rate. METHODS: We analyzed the results of 30 patients undergoing ITx and 49 patients treated with PTx. All patients were C-peptide-negative and suffered from hypoglycemia unawareness syndrome. Patients in the ITx group received a mean number of 12,349 (6,387-15,331) IEQ/kg/person administered percutaneously into the portal vein under local anesthesia and radiological control. The islet number was reached by 1-3 applications, as needed. In both groups, we evaluated glycated hemoglobin, insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia, and complications. We used the Mann Whitney test, Wilcoxon signed-rank test, and paired t-test for analysis. We also individually assessed the ITx outcomes for each patient according to recently suggested criteria established at the EPITA meeting in Igls. RESULTS: Most of the recipients showed a significant improvement in metabolic control one and two years after ITx, with a significant decrease in HbA1c, significant elevation of fasting and stimulated C-peptide, and a markedly significant reduction in insulin dose and the frequency of severe hypoglycemia. Seventeen percent of ITx recipients were temporarily insulin-independent. The results in the PTx group were comparable to those in the ITx group, with 73% graft survival and insulin independence in year 1, 68% 2 years and 55% 5 years after transplantation. There was a higher rate of complications related to the procedure in the PTx group. Severe hypoglycemia was eliminated in the majority of both ITx and PTx recipients. CONCLUSION: This report proves the successful initiation of pancreatic islet transplantation in a center with a well-established PTx program. ITx has been shown to be the method of choice for hypoglycemia unawareness syndrome, and may be considered for application in clinical practice if conservative options are exhausted.
- MeSH
- C-peptid krev MeSH
- diabetes mellitus 1. typu epidemiologie etiologie terapie MeSH
- dospělí MeSH
- hodnocení rizik MeSH
- hypoglykemie epidemiologie terapie MeSH
- krevní glukóza metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- následné studie MeSH
- přežívání štěpu MeSH
- retrospektivní studie MeSH
- syndrom MeSH
- transplantace Langerhansových ostrůvků * škodlivé účinky metody MeSH
- transplantace slinivky břišní * škodlivé účinky metody MeSH
- výběrové chování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
Transplantace Langerhansových ostrůvků (TxLO) byla v roce 2005 zavedena v Institutu klinické a experimentální medicíny (IKEM) jako potenciálně bezpečnější alternativa transplantace pankreatu (TxP), jež dosud představovala metodu volby u diabetiků 1. typu s konzervativně nezvládnutelným syndromem porušeného vnímání hypoglykemie. Cílem práce je porovnat schopnost obou metod eliminovat závažné hypoglykemie a další komplikace. Transplantaci Langerhansových ostrůvků od zavedení metody v roce 2005 do listopadu 2015 podstoupilo celkem 48 pacientů. Výsledky jsme hodnotili u 22 z nich, u kterých byl přítomen syndrom porušeného vnímání hypoglykemie. Počet transplantovaných ostrůvkových ekvivalentů činil 12 096 (6933−16 705) IEQ/kg, byly podány v místním znecitlivění perkutánně pod radiologickou kontrolou do portální žíly. TxP podstoupilo od roku 1996 44 pacientů. Hodnotili jsme glykovaný hemoglobin (HbA1c), dávky inzulinu, C-peptid lačný a stimulovaný, frekvenci těžkých hypoglykemií a komplikace. Mediány (interquartile range) jsme porovnávali pomocí Wilcoxonova rank testu. Po 1 i 2 letech od TxLO významně klesl HbA1c, byl měřitelný C-peptid, snížila se dávka inzulinu i frekvence hypoglykemií, 18 % příjemců bylo přechodně (maximálně 6 měsíců) bez potřeby inzulinu. Nejčastější komplikací bylo krvácení u 41 % pacientů. Jeden rok po TxP bylo 73 % pacientů bez potřeby inzulinu a hypoglykemií, 2 roky poté pak 68 % pacientů bez inzulinu a hypoglykemií, graftektomie proběhla u 20 % pacientů. Transplantace Langerhansových ostrůvků i transplantace pankreatu vedly k obnově sekrece inzulinu a eliminovaly těžké hypoglykemie. TxP vedla častěji k nezávislosti na inzulinu za cenu závažnějších komplikací. TxLO je méně invazivní metoda, kterou je potřeba dále rozvíjet.
Islet transplantation (ITx) started in 2005 in IKEM as a potentially safer alternative to pancreas transplantation (PTx), which so far had represented the method of choice in type-1 diabetic patients with conservatively intractable hypoglycemia unawareness syndrome. The aim of the study was to compare these two methods with regard to severe hypoglycemia elimination and to frequency of complications. Up to November 2015 a total number of 48 patients underwent ITx. The results from 22 patients with hypoglycemia unawareness were statistically analyzed. The mean number of transplanted islet equivalents was 12,096 (6,933−16,705) IEQ/kg administered percutaneously in local anesthesia under radiological control to the portal vein. 44 patients underwent PTx from 1996. We evaluated glycated hemoglobin(HbA1c), insulin dose, fasting and stimulated C-peptide, frequency of severe hypoglycemia and complications. Medians (interquartile range) were analyzed using the Wilcoxon signed-rank test. One and two years after ITx, HbA1c decreased, C-peptide became significantly positive, insulin dose and frequency of severe hypoglycemia decreased and 18 % of ITx recipients were temporarily insulin-independent. Bleeding was present in 41 % of patients. One year after PTx, 73 % of patients were insulin and hypoglycemia-free, after two years 68 % of patients were insulin and hypoglycemia-free; graftectomy occurred in 20 % of recipients. Both methods led to restoration of insulin secretion and severe hypoglycemia elimination. PTx made more recipients insulin-independent at the cost of serious complications.
- Klíčová slova
- syndrom porušeného vnímání hypoglykemie,
- MeSH
- C-peptid metabolismus MeSH
- diabetes mellitus 1. typu * chirurgie MeSH
- dospělí MeSH
- glykovaný hemoglobin analýza MeSH
- hypoglykemie diagnóza epidemiologie prevence a kontrola MeSH
- inzulin terapeutické užití MeSH
- lidé středního věku MeSH
- lidé MeSH
- odběr tkání a orgánů MeSH
- pilotní projekty MeSH
- pooperační komplikace MeSH
- prospektivní studie MeSH
- syndrom MeSH
- transplantace Langerhansových ostrůvků * metody MeSH
- transplantace slinivky břišní * metody MeSH
- výsledek terapie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
