IsletSwipe, a mobile platform for expert opinion exchange on islet graft images.

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.

Transplantation of Pancreatic Islets Into the Omentum Using a Biocompatible Plasma-Thrombin Gel: First Experience at the Institute for Clinical and Experimental Medicine in Prague.

BACKGROUND: Islet transplantation represents an established therapeutic option for people with type 1 diabetes who have hypoglycemia unawareness syndrome and frequent problematic hypoglycemic episodes when other methods comprising diabetes education and use of technological support fail. Because the current standard method of islet infusion into the liver has some limitations, novel approaches are under investigation. METHODS: We report our first results with 2 cases of islet transplantation into an omental pouch using a biocompatible plasma-fibrin gel. The recipients received 12,350 and 5,350 islet equivalents per kilogram that were mixed with autologous plasma, seeded during a laparoscopic procedure on the omentum, overlaid with human thrombin solution, and fixed by flapping the omentum over. RESULTS: During a 9-month follow-up, neither patient experienced any moderate or severe hypoglycemia. Their glucose control significantly improved, insulin dose decreased by approximately 50%, and C-peptide at 1 year was 0.22 and 0.14 pmol/mL, respectively. The postoperative course was uneventful, but C-peptide production in the first patient progressively declined at 1 year and hypoglycemic episodes recurred. CONCLUSIONS: Though the results for these first 2 cases are not fully satisfactory, we have demonstrated the feasibility, safety, and ability of this novel method to restore insulin production. Further refinements to improve immediate islet survival seem necessary.

The Potential of Pancreatic Organoids for Diabetes Research and Therapy.

The success of clinical transplantation of pancreas or isolated pancreatic islets supports the concept of cell-based cure for diabetes. One limitation is the shortage of cadaver human pancreata. The demand-supply gap could potentially be bridged by harnessing the self-renewal capacity of stem cells. Pluripotent stem cells and adult pancreatic stem cells have been explored as possible cell sources. Recently, a system for long-term culture of proposed adult pancreatic stem cells in a form of organoids was developed. Generated organoids partially mimic the architecture and cell-type composition of pancreatic tissue. Here, we review the attempts over the past decade, to utilize the organoid cell culture principles in order to identify, expand, and differentiate the adult pancreatic stem cells from different compartments of mouse and human pancreata. The development of the culture conditions, effects of specific growth factors and small molecules is discussed. The potential utility of the adult pancreatic stem cells is considered in the context of other cell sources.

Characterization of large in-frame von Willebrand factor deletions highlights differing pathogenic mechanisms.

Copy number variation (CNV) is known to cause all von Willebrand disease (VWD) types, although the associated pathogenic mechanisms involved have not been extensively studied. Notably, in-frame CNV provides a unique opportunity to investigate how specific von Willebrand factor (VWF) domains influence the processing and packaging of the protein. Using multiplex ligation-dependent probe amplification, this study determined the extent to which CNV contributed to VWD in the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease cohort, highlighting in-frame deletions of exons 3, 4-5, 32-34, and 33-34. Heterozygous in vitro recombinant VWF expression demonstrated that, although deletion of exons 3, 32-34, and 33-34 all resulted in significant reductions in total VWF (P < .0001, P < .001, and P < .01, respectively), only deletion of exons 3 and 32-34 had a significant impact on VWF secretion (P < .0001). High-resolution microscopy of heterozygous and homozygous deletions confirmed these observations, indicating that deletion of exons 3 and 32-34 severely impaired pseudo-Weibel-Palade body (WPB) formation, whereas deletion of exons 33-34 did not, with this variant still exhibiting pseudo-WPB formation similar to wild-type VWF. In-frame deletions in VWD, therefore, contribute to pathogenesis via moderate or severe defects in VWF biosynthesis and secretion.

Synthetic mRNA is a more reliable tool for the delivery of DNA-targeting proteins into the cell nucleus than fusion with a protein transduction domain.

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.

Benefits of Islet Transplantation as an Alternative to Pancreas Transplantation: Retrospective Study of More Than 10 Ten Years of Experience in a Single Center.

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.

[Islet transplantation as a treatment for hypoglycemia unawareness syndrome. Evaluation of the pilot program and comparison with pancreas transplantation]. / Transplantace Langerhansových ostruvku v lécbe syndromu poruseného vnímání hypoglykemie. Vyhodnocení pilotního programu a porovnání s transplantací pankreatu.

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 (6,93316,705) 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.

Combining Donor Characteristics with Immunohistological Data Improves the Prediction of Islet Isolation Success.

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.

Automated Analysis of Microscopic Images of Isolated Pancreatic Islets.

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.

Conditional knockout of integrin α2ß1 in murine megakaryocytes leads to reduced mean platelet volume.

We have engineered a transgenic mouse on a C57BL/6 background that bears a floxed Itga2 gene. The crossing of this mouse strain to transgenic mice expressing Cre recombinase driven by the megakaryocyte (MK)-specific Pf4 promoter permits the conditional knockout of Itga2 in the MK/platelet lineage. Mice lacking MK α2ß1 develop normally, are fertile, and like their systemic α2ß1 knockout counterparts, exhibit defective adhesion to and aggregation induced by soluble type I collagen and a delayed onset to low dose fibrillar collagen-induced aggregation, results consistent with blockade or loss of platelet α2ß1. At the same time, we observed a significant reduction in mean platelet volume, which is consistent with the reported role of α2ß1 in MK maturation and proplatelet formation in vivo. This transgenic mouse strain bearing a floxed Itga2 gene will prove valuable to distinguish in vivo the temporal and spatial contributions of α2 integrin in selected cell types.

[Islet transplantation for treatment of type-1 diabetes mellitus]. / Lécba diabetu transplantací izolovaných Langerhansových ostruvku.

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.

The impact of bleeding history, von Willebrand factor and PFA-100(®) on the diagnosis of type 1 von Willebrand disease: results from the European study MCMDM-1VWD.

The relationships between the Platelet Function Analyzer (PFA)-100 and von Willebrand factor (VWF) levels and bleeding score (BS) were evaluated within a multicentre project on Molecular and Clinical Markers for the Diagnosis and Management of type 1 von Willebrand disease (MCMDM-1VWD). PFA-100 closure time, either with epinephrine (EPI) or adenosine diphosphate (ADP)-cartridges, was measured in 107 index cases, 105 affected and 71 unaffected family members, and 79 healthy controls. By regression analysis VWF levels were strongly related to both closure times, with a non-linear progression. In a multiple stepwise regression model, age- and sex-adjusted PFA-100 ADP and VWF ristocetin cofactor activity (VWF:RCo) were independently associated with BS. Most of the variation of BS was predicted by PFA-100 ADP and VWF:RCo alone. In the subgroup of patients with subtle abnormalities of the multimeric pattern, VWF was invariably reduced and closure time prolonged in almost all of them. Neither PFA-100 ADP nor EPI closure times appeared to significantly improve the diagnostic capability of VWF antigen (VWF:Ag) measurement. Thus, in an unselected population a normal PFA-100 would be useful to exclude VWD, but whether it could replace the more specific VWF assay in patients with significant mucocutaneous bleeding symptoms remains to be investigated prospectively.

Factor XI gene mutations in factor XI deficient patients of the Czech Republic.

Factor XI (FXI) deficiency is an autosomal inherited coagulation disorder characterized by bleeding symptoms mainly associated with injury or surgery. Although most of the FXI gene mutations in Ashkenazi Jews are represented by the Glu117stop or Phe283Leu mutations, considerable genetic heterogeneity has been reported in other populations. We report here the genotypic characterization of four families with severe inherited FXI deficiency from the Czech Republic. Seven different gene mutations (three novel) were identified, thus, excluding the existence of a major founder effect in this population. Interestingly, both Glu117stop and Phe283Leu were detected once, further demonstrating the occurrence of these mutations also outside the Jewish populations. In conclusion, we confirm that FXI deficiency in non-Jewish populations is because of different gene mutations; however, the presence of the Glu117stop and Phe283Leu mutations suggests that genetic testing in FXI-deficient patients can start with these two point mutations.

Identification of type 1 von Willebrand disease patients with reduced von Willebrand factor survival by assay of the VWF propeptide in the European study: molecular and clinical markers for the diagnosis and management of type 1 VWD (MCMDM-1VWD).

The decreased survival of von Willebrand factor (VWF) in plasma has been implicated as a mechanism in a subset of type 1 von Willebrand disease (VWD) patients. We have previously reported that the ratio of plasma levels of VWF and its propeptide (VWFpp) can be used to identify patients with reduced VWF survival. In this study, we report the assay of VWFpp and VWF:Ag in 19 individuals recruited from 6 European centers within the MCMDM-1VWD study. Eight individuals had a VWF:Ag level less than 30 IU/dL. Seven of these patients had a robust desmopressin response and significantly reduced VWF half-life that was predicted by a markedly increased steady-state plasma VWFpp/VWF:Ag ratio. VWF mutations previously associated with reduced VWF survival were identified in each of the 7 individuals. Thus, a substantially increased ratio of steady-state VWFpp/VWF:Ag predicted a reduced VWF half-life in patients with markedly decreased VWF:Ag levels. These data indicate that a reduced VWF survival is found in a subpopulation of patients with type 1 VWD. The systematic assay of both plasma VWF and the VWF propeptide in moderately severe type 1 VWD patients may identify patients with a reduced VWF survival phenotype.

Response to desmopressin is influenced by the genotype and phenotype in type 1 von Willebrand disease (VWD): results from the European Study MCMDM-1VWD.

We have prospectively evaluated the biologic response to desmopressin in 77 patients with type 1 von Willebrand disease (VWD) enrolled within the Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD project. Complete response to desmopressin was defined as an increase of both ristocetin cofactor activity (VWF:RCo) and factor VIII coagulant activity (FVIII:C) to 50 IU/dL or higher and partial response as VWF:RCo or FVIII:C lower than 50 IU/dL after infusion, but at least 3-fold the basal level. Complete response was observed in 83% of patients; partial in 13%; and no response in 4%. Patients with some abnormality of VWF multimeric pattern had significantly lower basal FVIII:C and VWF, lower VWF:RCo/Ag ratio, and less complete responses to desmopressin than patients with a normal multimeric pattern (P=.002). Patients with mutations at codons 1130 and 1205 in the D'-D3 domain had the greatest relative increase, but shortest FVIII and VWF half-lives after infusion. Most partial and nonresponsive patients had mutations in the A1-A3 domains. Response to desmopressin in these VWD patients seemed to be associated with the location of the causative mutation. The presence of subtle multimeric abnormalities did not hamper potential clinically useful responses, as in typical type 1 VWD.

The Modifier of hemostasis (Mh) locus on chromosome 4 controls in vivo hemostasis of Gp6-/- mice.

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 x 1/SvJ x C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6(-/-) mice is uniform and is not affected by genetic background. However, the same Gp6(-/-) mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide single-nucleotide polymorphism scan revealed the most significant linkage to a single locus (8 megabases) on chromosome 4 (logarithm of the odds [LOD] score = 6.9, P < .0001) that we designate Modifier of hemostasis (Mh). Our results indicate that one or more modifier genes in Mh control the extent to which in vivo platelet thrombus formation is disrupted by the absence of platelet GPVI.

Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD).

Type 1 von Willebrand disease (VWD) is characterized by a personal and family history of bleeding coincident with reduced levels of normal plasma von Willebrand factor (VWF). The molecular basis of the disorder is poorly understood. The aims of this study were to determine phenotype and genotype and their relationship in patients historically diagnosed with type 1 VWD. Families were recruited in 9 European countries based on previous type 1 VWD diagnosis. Bleeding symptoms were recorded, plasma phenotype analyzed, and VWF mutation analysis performed in all index cases (ICs). Phenotypic and molecular analysis stratified patients into those with or without phenotypes suggestive of qualitative VWF defects (abnormal multimers) and with or without mutations. A total of 105 of 150 ICs (70%) had mutations identified. A subgroup with abnormal multimers (38% of ICs, 57 of 150) showed a high prevalence of VWF gene mutations (95% of ICs, 54 of 57), whereas in those with qualitatively normal VWF, fewer mutations were identified (55% of ICs, 51 of 93). About one third of the type 1 VWD cases recruited could be reconsidered as type 2. The remaining group could be considered "true" type 1 VWD, although mutations were found in only 55%.

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination.

BACKGROUND: We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. METHODS: Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. RESULTS: The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). CONCLUSIONS: Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.