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
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
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
PURPOSE: An artificial site for cell or pancreatic islet transplantation can be created using a polymeric scaffold, even though it suffers subcutaneously from improper vascularisation. A sufficient blood supply is crucial for graft survival and function and can be enhanced by transplantation of mesenchymal stem cells (MSCs). The purpose of this study was to assess the effect of syngeneic MSCs on neoangiogenesis and cell engraftment in an artificial site by multimodal imaging. PROCEDURES: MSCs expressing a gene for luciferase were injected into the artificial subcutaneous site 7 days after scaffold implantation. MRI experiments (anatomical and dynamic contrast-enhanced images) were performed on a 4.7-T scanner using gradient echo sequences. Bioluminescent images were acquired on an IVIS Lumina optical imager. Longitudinal examination was performed for 2 months, and one animal was monitored for 16 months. RESULTS: We confirmed the long-term presence (lasting more than 16 months) of viable donor cells inside the scaffolds using bioluminescence imaging with an optical signal peak appearing on day 3 after MSC implantation. When compared to controls, the tissue perfusion and vessel permeability in the scaffolds were significantly improved at the site with MSCs with a maximal peak on day 9 after MSC transplantation. CONCLUSIONS: Our data suggest that the maximal signal obtained by bioluminescence and magnetic resonance imaging from an artificially created site between 3 and 9 days after MSC transplantation can predict the optimal time range for subsequent cellular or tissue transplantation, including pancreatic islets.
- MeSH
- kontrastní látky MeSH
- luminiscenční měření MeSH
- magnetická rezonanční tomografie MeSH
- mezenchymální kmenové buňky cytologie MeSH
- multimodální zobrazování * MeSH
- potkani inbrední LEW MeSH
- regionální krevní průtok fyziologie MeSH
- reprodukovatelnost výsledků MeSH
- tkáňové podpůrné struktury MeSH
- transplantace mezenchymálních kmenových buněk * MeSH
- umělé buňky * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
Clinical islet transplantation programs rely on the capacities of individual centers to quantify isolated islets. Current computer-assisted methods require input from human operators. Here we describe two machine learning algorithms for islet quantification: the trainable islet algorithm (TIA) and the nontrainable purity algorithm (NPA). These algorithms automatically segment pancreatic islets and exocrine tissue on microscopic images in order to count individual islets and calculate islet volume and purity. References for islet counts and volumes were generated by the fully manual segmentation (FMS) method, which was validated against the internal DNA standard. References for islet purity were generated via the expert visual assessment (EVA) method, which was validated against the FMS method. The TIA is intended to automatically evaluate micrographs of isolated islets from future donors after being trained on micrographs from a limited number of past donors. Its training ability was first evaluated on 46 images from four donors. The pixel-to-pixel comparison, binary statistics, and islet DNA concentration indicated that the TIA was successfully trained, regardless of the color differences of the original images. Next, the TIA trained on the four donors was validated on an additional 36 images from nine independent donors. The TIA was fast (67 s/image), correlated very well with the FMS method (R2=1.00 and 0.92 for islet volume and islet count, respectively), and had small REs (0.06 and 0.07 for islet volume and islet count, respectively). Validation of the NPA against the EVA method using 70 images from 12 donors revealed that the NPA had a reasonable speed (69 s/image), had an acceptable RE (0.14), and correlated well with the EVA method (R2=0.88). Our results demonstrate that a fully automated analysis of clinical-grade micrographs of isolated pancreatic islets is feasible. The algorithms described herein will be freely available as a Fiji platform plugin.
- MeSH
- algoritmy MeSH
- automatizace MeSH
- krysa rodu rattus MeSH
- Langerhansovy ostrůvky * cytologie MeSH
- lidé MeSH
- počítačové zpracování obrazu * MeSH
- potkani Wistar MeSH
- strojové učení MeSH
- transplantace Langerhansových ostrůvků * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
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/m(2) (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.
- MeSH
- dárci tkání statistika a číselné údaje MeSH
- délka pobytu statistika a číselné údaje MeSH
- diabetes mellitus 1. typu chirurgie MeSH
- edém epidemiologie MeSH
- hospitalizace MeSH
- imunohistochemie MeSH
- index tělesné hmotnosti MeSH
- inzulin metabolismus MeSH
- Langerhansovy ostrůvky metabolismus chirurgie MeSH
- lidé MeSH
- pankreas MeSH
- retrospektivní studie MeSH
- studená ischemie statistika a číselné údaje MeSH
- transplantace Langerhansových ostrůvků metody MeSH
- vazokonstriktory terapeutické užití MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
