• MeSH
• diabetes mellitus 1. typu * chirurgie   MeSH
• krevní glukóza * MeSH
• lidé MeSH
• selfmonitoring glykemie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH

Clostridial collagenases are essential biotechnological tissue dissociation agents owing to their ability to cleave different types of collagen. Standardization of collagenase-based protocols has been hampered by impurities in products manufactured from Clostridium histolyticum. To enhance the purification process, we produced recombinant collagenase classes G and H, taking advantage of the Escherichia coli expression system. The respective gene sequences were derived from C. histolyticum and modified by addition of a C-terminal polyhistidine tag. Harvested bacteria were lysed and the collagenase protein was affinity purified using a His-tag column. The purity, identity, integrity of the eluted collagenases G and H were determined by SDS electrophoresis and Western blot. The proteolytic activity of the collagenase G and H blend (rColGH) was determined by the standard FALGPA assay. The tissue dissociation activity was verified using a standardized method for isolation of rat pancreatic islets. Biocompatibility of the blend was validated by a standardized viability assay on the isolated islets. Two batches of rColGH were produced and compared to a commercially available collagenase. Based on our results, we conclude that rColGH is a functional and non-toxic novel recombinant collagenase worth further characterization and blend optimization in order to make it a competitive commercial product.

• MeSH
• Clostridium MeSH
• kolagenasy * MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• buněčná diferenciace MeSH
• diabetes mellitus * terapie   MeSH
• lidé MeSH
• organoidy MeSH
• pankreas MeSH
• pluripotentní kmenové buňky * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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.

• MeSH
• lidé MeSH
• variabilita počtu kopií segmentů DNA MeSH
• von Willebrandova nemoc, typ 1 * MeSH
• von Willebrandova nemoc * diagnóza   genetika   MeSH
• von Willebrandův faktor genetika   MeSH
• Weibel-Paladeho granula MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Reprogramming of non-endocrine pancreatic cells into insulin-producing cells represents a promising therapeutic approach for the restoration of endogenous insulin production in diabetic patients. In this paper, we report that human organoid cells derived from the pancreatic tissue can be reprogrammed into the insulin-producing cells (IPCs) by the combination of in vitro transcribed modified mRNA encoding transcription factor neurogenin 3 and small molecules modulating the epigenetic state and signalling pathways. Upon the reprogramming, IPCs formed 4.6 ± 1.2 % of the total cells and expressed typical markers (insulin, glucokinase, ABCC8, KCNJ11, SLC2A2, SLC30A8) and transcription factors (PDX1, NEUROD1, MAFA, NKX2.2, NKX6.1, PAX4, PAX6) needed for the proper function of pancreatic β-cells. Additionally, we have revealed a positive effect of ALK5 inhibitor RepSox on the overall reprogramming efficiency. However, the reprogrammed IPCs possessed only a partial insulin-secretory capacity, as they were not able to respond to the changes in the extracellular glucose concentration by increasing insulin secretion. Based on the achieved results we conclude that due to the incomplete reprogramming, the IPCs have immature character and only partial properties of native human β-cells.

• MeSH
• antigen AC133 metabolismus   MeSH
• beta-buňky cytologie   účinky léků   MeSH
• dospělí MeSH
• genetická transkripce účinky léků   MeSH
• inzulin biosyntéza   MeSH
• knihovny malých molekul farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• organoidy cytologie   MeSH
• přeprogramování buněk účinky léků   genetika   MeSH
• proliferace buněk MeSH
• proteiny nervové tkáně genetika   metabolismus   MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Úvod: Agonista receptoru pro GLP1 lixisenatid prokázal svou účinnost v četných klinických studiích, avšak údaje o jeho účinnosti z reálného života jsou omezené. Cíl: Popis účinnosti a bezpečnosti lixisenatidu v běžné klinické praxi v České republice a Slovenské republice, jak jsou zaznamenány v Observační studii na podkladě údajů z registru. Metody: Mnohonárodní multicentrický, observační, neintervenční, 6měsíční prospektivní produktový registr pacientů s diabetem 2. typu nad 18 let, kteří začínali léčbu lixisenatidem. Na základě informovaného písemného souhlasu byli pacienti do tohoto registru zařazeni v období od 1. května 2013 do 31. prosince 2015. Hodnocení byla provedena na začátku studie a dále po 3 a 6 měsících léčby lixisenatidem. Primárním cílem studie bylo dosažení zásadní změny v hodnotě glykovaného hemoglobinu (HbA1c) v období od počátku léčby do 6. měsíce po zavedení lixisenatidu. Studie byla schválena příslušnými etickými komisemi a proběhla v souladu s Helsinskou deklarací. Informovaný souhlas byl získán od všech pacientů před jejich zařazením do studie. Výsledky: Do studie bylo zařazeno celkem 772 pacientů (z toho 51,4 % mužů) v průměrném věku 56,7 (± 9,3) let, s průměrnou dobou trvání diabetu 7,7 (± 5,5) let, s průměrnou dobou léčby perorálními antidiabetiky (PAD) 6,8 (± 4,9) let a indexem tělesné hmotnosti (BMI) 37,6 (± 5,9) kg/m2. Celkem 93,6 % účastníků studie bylo obézních, 86,3 % se léčilo pro hypertenzi a u 76,0 % účastníků probíhala léčba dyslipidemie. Celkem 96,1 % pacientů dokončilo 6měsíční terapii. Lixisenatid významně snížil hodnotu HbA1c (pokles o 9,7 ± 14,4 mmol/mol [3,1 ± 0,2 % DCCT] po 6 měsících v populaci per protokol), a tělesnou hmotnost (pokles o 3,5 ± 5,4 kg). Nejlépe reagovali na léčbu mladší pacienti s vyšším BMI a s kratší dobou trvání diabetu. Celkový bezpečnostní profil lixisenatidu byl v rámci studie uspokojivý. Nejčastější nežádoucí účinky gastrointestinální. V průběhu celé studie nebyla hlášena žádná epizoda těžké hypoglykemie. Závěr: U skupiny pacientů s diabetem 2. typu v podmínkách běžného života se po 6měsíční terapii při užívání agonisty receptoru pro GLP1 lixisenatidu 1krát denně významně zlepšila kompenzace diabetu a snížila tělesná hmotnost, aniž by se zvýšilo riziko symptomatické a/nebo těžké hypoglykemie. Financování studie: Sanofi Česká republika

Introduction: GLP1 receptor agonist lixisenatide has demonstrated its efficacy in numerous clinical trials, nevertheless its real-life effectiveness data is limited. Aim: To describe effectiveness and safety of lixisenatide in routine clinical practice in the Czech Republic and the Slovak Republic, as recorded by the Registry-Based Observational Study. Methods: Multinational, multicenter, observational, non-interventional, 6-month prospective product registry of patients with type 2 diabetes mellitus aged > 18 years who were initiating therapy with lixisenatide. Patients were enrolled into this registry, provided written informed consent, between 1 May 2013 and 31 December 2015. Evaluations were performed at baseline and after 3 and 6 months of lixisenatide treatment. The primary objective of the study was the absolute change in glycated hemoglobin (HbA1c) from baseline to month 6 after lixisenatide initiation. The study was approved by responsible ethics committees and performed in accordance with the Helsinki Declaration. Informed consent was obtained from all patients before enrolment in the study. Results: Overall 772 eligible patients (51.4 % males), mean age 56.7 (± 9.3) years, with mean diabetes duration 7.7 (± 5.5) years, mean duration of treatment with oral antidiabetic drugs 6.8 (± 4.9) years, and body mass index 37.6 (± 5.9) kg/m2 were enrolled in the study. Overall, 93.6 % were obese, 86.3 % subject were treated for hypertension, and 76.0 % for dyslipidemia. In total 96.1 % of patients completed the 6 months’ therapy. Lixisenatide significantly reduced HbA1c (decrease by 9.7 ± 14.4 mmol/mol [3.1 ± 0.2 % DCCT] after 6 months in per protocol population), and body weight (decrease by 3.5 ± 5.4 kg). The best responders to the treatment were younger patients with higher BMI, who had a shorter duration of diabetes. Overall safety profile of lixisenatide was satisfactory in the study. The most frequent adverse events were functional disorders affecting the gastrointestinal system. There was no episode of severe hypoglycemia reported throughout the study. Conclusion: In a real-life practice cohort of patients with type 2 diabetes mellitus 6 months treatment with once-daily GLP1 receptor agonist lixisenatide significantly improved glucose control and decreased body weight without increasing the risk of symptomatic and/or severe hypoglycemia risk. Funding: Sanofi Czech Republic.

• Klíčová slova
• lixisenatid,
• MeSH
• diabetes mellitus 2. typu * farmakoterapie   metabolismus   MeSH
• dospělí MeSH
• glykovaný hemoglobin metabolismus   MeSH
• hmotnostní úbytek * účinky léků   MeSH
• hypoglykemie epidemiologie   MeSH
• hypoglykemika škodlivé účinky   terapeutické užití   MeSH
• index tělesné hmotnosti MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• peptidy škodlivé účinky   terapeutické užití   MeSH
• prospektivní studie MeSH
• receptor pro glukagonu podobný peptid 1 MeSH
• senioři MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• multicentrická studie MeSH
• pozorovací studie 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: 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

Direct reprogramming of pancreatic nonendocrine cells into insulin-producing β-cells represents a promising approach for the treatment of insulin-dependent diabetes. However, its clinical application is limited by the potential for insertional mutagenesis associated with the viral vectors currently used for cell reprogramming. With the aim of developing a nonintegrative reprogramming strategy for derivation of insulin-producing cells, here, we evaluated a new approach utilizing synthetic messenger RNAs encoding reprogramming transcription factors. Administration of synthetic mRNAs encoding three key transcription regulators of β-cell differentiation-Pdx1, Neurogenin3, and MafA-efficiently reprogrammed the pancreatic exocrine cells into insulin-producing cells. In addition to the insulin genes expression, the synthetic mRNAs also induced the expressions of genes important for proper pancreatic β-cell function, including Sur1, Kir6.2, Pcsk1, and Pcsk2. Pretreating cells with the chromatin-modifying agent 5-Aza-2'-deoxycytidine further enhanced reprogramming efficiency, increasing the proportion of insulin-producing cells from 3.5 ± 0.9 to 14.3 ± 1.9% (n = 4). Moreover, 5-Aza-2'-deoxycytidine pretreatment enabled the reprogrammed cells to respond to glucose challenge with increased insulin secretion. In conclusion, our results support that the reprogramming of pancreatic exocrine cells into insulin-producing cells, induced by synthetic mRNAs encoding pancreatic transcription factors, represents a promising approach for cell-based diabetes therapy.

• Publikační typ
• časopisecké články MeSH