PubMed Health⌕ Search

Biomedical subjects

Timo Otonkoski

Publications and source records attributed to Timo Otonkoski.

At least 19 recordsLinked to original sources
PubMed2006

Maturation of in vitro-generated human islets after transplantation in nude mice.

The long-term function of human pancreatic islet grafts may depend on the neogenesis of beta cells from epithelial precursors within the grafted tissue. We have developed an in vitro model for human islet neogenesis. In this study, we have investigated the morphological signs of maturation in cultivated human islet buds (CHIBs) before and after transplantation. Clusterin is a molecule associated with beta-cell differentiation in rodents. In adult human islets, clusterin expression was located only in alpha- and PP-cells, but in CHIBs and human fetal islets, it was distributed in all four types of endocrine cells. Some immature endocrine cells in the CHIBs co-expressed insulin and glucagon. After transplantation, CHIBs became mature with one type of hormone per endocrine cell, and clusterin expression became restricted in alpha-cells. Cells co-expressing endocrine markers and cytokeratin 19, as a sign of ductal to endocrine cell transition, were frequently detected in both fresh islets and CHIBs after transplantation. We conclude that clusterin may be involved in the development of islets, and the in vitro-derived islets become mature after transplantation into nude mice. Ductal cell differentiation into endocrine cells may be an important factor in sustaining the long-term function of islet transplants.

Animals↗
PubMed2006

Rapid genetic analysis in congenital hyperinsulinism.

BACKGROUND: In severe, medically unresponsive congenital hyperinsulinism (CHI), the histological differentiation of focal versus diffuse disease is vital, since the surgical management is completely different. Genetic analysis may help in the differential diagnosis, as focal CHI is associated with a paternal germline ABCC8 or KCNJ11 mutation and a focal loss of maternal chromosome 11p15, whereas a maternal mutation, or homozygous/compound heterozygous ABCC8 and KCNJ11 mutations predict diffuse-type disease. However, genotyping usually takes too long to be helpful in the absence of a founder mutation. METHODS: In 4 patients, a rapid genetic analysis of the ABBC8 and KCNJ11 genes was performed within 2 weeks on request prior to the decision of pancreatic surgery. RESULTS: Two patients had no mutations, rendering the genetic analysis non-informative. Peroperative multiple biopsies showed diffuse disease. One patient had a paternal KCNJ11 mutation and focal disease confirmed by positron emission tomography scan and biopsies. One patient had a de novo heterozygous ABBC8 mutation and unexplained diffuse disease confirmed by positron emission tomography scan and biopsies. CONCLUSION: A rapid analysis of the entire ABBC8 and KCNJ11 genes should not stand alone in the preoperative assessment of patients with CHI, except for the case of maternal, or homozygous/compound heterozygous disease-causing mutations.

ATP-Binding Cassette Transporters↗
PubMed2006

N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible.

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.

Antibodies↗
PubMed2006

Distinct differentiation characteristics of individual human embryonic stem cell lines.

BACKGROUND: Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. RESULTS: The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. CONCLUSION: hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state.

Animals↗
PubMed2006

Downregulation of EGF receptor signaling in pancreatic islets causes diabetes due to impaired postnatal beta-cell growth.

Epidermal growth factor receptor (EGF-R) signaling is essential for proper fetal development and growth of pancreatic islets, and there is also evidence for its involvement in beta-cell signal transduction in the adult. To study the functional roles of EGF-R in beta-cell physiology in postnatal life, we have generated transgenic mice that carry a mutated EGF-R under the pancreatic duodenal homeobox-1 promoter (E1-DN mice). The transgene was expressed in islet beta- and delta-cells but not in alpha-cells, as expected, and it resulted in an approximately 40% reduction in pancreatic EGF-R, extracellular signal-related kinase, and Akt phosphorylation. Homozygous E1-DN mice were overtly diabetic after the age of 2 weeks. The hyperglycemia was more pronounced in male than in female mice. The relative beta-cell surface area of E1-DN mice was highly reduced at the age of 2 months, while alpha-cell surface area was not changed. This defect was essentially postnatal, since the differences in beta-cell area of newborn mice were much smaller. An apparent explanation for this is impaired postnatal beta-cell proliferation; the normal surge of beta-cell proliferation during 2 weeks after birth was totally abolished in the transgenic mice. Heterozygous E1-DN mice were glucose intolerant in intraperitoneal glucose tests. This was associated with a reduced insulin response. However, downregulation of EGF-R signaling had no influence on the insulinotropic effect of glucagon-like peptide-1 analog exendin-4. In summary, our results show that even a modest attenuation of EGF-R signaling leads to a severe defect in postnatal growth of the beta-cells, which leads to the development of diabetes.

Animals↗
PubMed2006

Noninvasive diagnosis of focal hyperinsulinism of infancy with [18F]-DOPA positron emission tomography.

Congenital hyperinsulinism of infancy (CHI) is characterized by severe hypoglycemia due to dysregulated insulin secretion, associated with either focal or diffuse pathology of the endocrine pancreas. The focal condition is caused by a paternally inherited mutation in one of the genes encoding the subunits of the beta-cell ATP-sensitive potassium channel (SUR1/ABCC8 or Kir6.2/KCNJ11) and somatic loss of maternal 11p15 alleles within the affected area. Until now, preoperative diagnostics have relied on technically demanding and invasive catheterization techniques. We evaluated the utility of fluorine-18 l-3,4-dihydroxyphenylalanine ([(18)F]-DOPA) positron emission tomography (PET) to identify focal pancreatic lesions in 14 CHI patients, 11 of which carried mutations in the ABCC8 gene (age 1-42 months). To reduce bias in PET image interpretation, quantitative means for evaluation of pancreatic [(18)F]-DOPA uptake were established. Five patients had a visually apparent focal accumulation of [(18)F]-DOPA and standardized uptake value (SUV) >50% higher (mean 1.8-fold) than the maximum SUV of the unaffected part of the pancreas. When these patients were operated on, a focus of 4-5 x 5-8 mm matching with the PET scan was found, and all were normoglycemic after resection of the focus. The remaining nine patients had diffuse accumulation of [(18)F]-DOPA in the pancreas (SUV ratio <1.5). Diffuse histology was verified in four of these, and pancreatic catheterization was consistent with diffuse pathology in four cases. In conclusion, [(18)F]-DOPA PET is a promising noninvasive method for the identification and localization of the focal form of CHI.

Child, Preschool↗
PubMed2005

Gene expression signatures of seven individual human embryonic stem cell lines.

Identification of molecular components that define a pluripotent human embryonic stem cell (hESC) provides the basis for understanding the molecular mechanisms regulating the maintenance of pluripotency and induction of differentiation. We compared the gene expression profiles of seven genetically independent hESC lines with those of nonlineage-differentiated cells derived from each line. A total of 8,464 transcripts were expressed in all hESC lines. More than 45% of them have no yet-known biological function, which indicates that a high number of unknown factors contribute to hESC pluripotency. Among these 8,464 transcripts, 280 genes were specific for hESCs and 219 genes were more than twofold differentially expressed in all hESC lines compared with nonlineage-differentiated cells. They represent genes implicated in the maintenance of pluripotency and those involved in early differentiation. The chromosomal distribution of these hESC-enriched genes showed over-representation in chromosome 19 and under-representation in chromosome 18. Although the overall gene expression profiles of the seven hESC lines were markedly similar, each line also had a subset of differentially expressed genes reflecting their genetic variation and possibly preferential differentiation potential. Limited overlap between gene expression profiles illustrates the importance of cross-validation of results between different ESC lines.

Cell Differentiation↗
PubMed2005

Over- and underdosage of SOX3 is associated with infundibular hypoplasia and hypopituitarism.

Duplications of Xq26-27 have been implicated in the etiology of X-linked hypopituitarism associated with mental retardation (MR). Additionally, an expansion of a polyalanine tract (by 11 alanines) within the transcription factor SOX3 (Xq27.1) has been reported in patients with growth hormone deficiency and variable learning difficulties. We report a submicroscopic duplication of Xq27.1, the smallest reported to date (685.6 kb), in two siblings with variable hypopituitarism, callosal abnormalities, anterior pituitary hypoplasia (APH), an ectopic posterior pituitary (EPP), and an absent infundibulum. This duplication contains SOX3 and sequences corresponding to two transcripts of unknown function; only Sox3 is expressed in the infundibulum in mice. Next, we identified a novel seven-alanine expansion within a polyalanine tract in SOX3 in a family with panhypopituitarism in three male siblings with an absent infundibulum, severe APH, and EPP. This mutation led to reduced transcriptional activity, with impaired nuclear localization of the mutant protein. We also identified a novel polymorphism (A43T) in SOX3 in another child with hypopituitarism. In contrast to findings in previous studies, there was no evidence of MR or learning difficulties in our patients. We conclude that both over- and underdosage of SOX3 are associated with similar phenotypes, consisting of infundibular hypoplasia and hypopituitarism but not necessarily MR.

Adolescent↗
PubMed2005

Stem cells in the treatment of diabetes.

Clinical islet transplantation trials based on cadaveric allogenic islets have demonstrated that it is indeed possible to restore near-physiological insulin secretion capacity in a type 1 diabetic patient through transplantation of insulin-producing cells. In order to develop this form of therapy to become available for the vast majority of patients with diabetes, new sources of transplantable insulin-producing cells need to be identified. Stem cells provide the best potential to achieve this goal. Controversial results have been presented concerning the existence and nature of pancreatic islet stem or precursor cells. An increasing body of evidence suggests that the pancreatic and hepatic cell types (hepatocytes, islet, acinar and ductal cells) have remarkable plasticity and can de- and trans-differentiate into each other under appropriate conditions. Elucidation of the molecular mechanisms regulating these processes could lead to clinically applicable ways of either inducing pancreatic islet regeneration in situ or to expanding the insulin-producing cells in vitro for transplantation. The emergence of human embryonic stem cells has led to an active area of research aiming to achieve targeted differentiation of these cells into a safely transplantable beta-like cell. After initial excitement, it appears that much basic research is still required before this goal could be achieved.

Animals↗
PubMed2005

Lack of association of PAX4 gene with type 1 diabetes in the Finnish and Hungarian populations.

We aimed to assess the possible contribution of the PAX4 transcription factor gene to the genetic background of type 1 diabetes. We analyzed four coding polymorphisms of the PAX4 gene in 498 cases with type 1 diabetes and 825 control subjects from Finland and Hungary. All patients were diagnosed under the age of 15 years according to the World Health Organization criteria. All four PAX4 variants (three in exon 9 and one in exon 3) were genotyped using DNA sequencing. In addition, all Finnish subjects were typed for HLA DR-DQ, insulin gene (-23) HphI, and CTLA4 CT60 polymorphisms. The +1,168 C/A coding variant of PAX4 was found to be polymorphic in both populations (P321H, rs712701). No difference was observed in the genotype frequencies between cases and control subjects, nor was any disease association detected when patients were stratified according to age at diagnosis, sex, HLA, insulin gene, or CTLA4 genotypes. Our data indicate that the +1,168 C/A variant of PAX4 gene does not play any essential role in genetic type 1 diabetes susceptibility. The strong coherence between the datasets of the two ethnic groups studied with highly contrasting disease incidence, socioeconomic characteristics, and profoundly diverse environment emphasizes the impact of this finding.

Adolescent↗
PubMed2004

Transcription factor GATA-6 is expressed in the endocrine and GATA-4 in the exocrine pancreas.

GATA-4 and GATA-6 are zinc finger transcription factors that regulate gene expression, differentiation, and cell proliferation in various tissues. These factors have been implicated in the development of endodermal derivatives, including epithelial cells in the yolk sac, lung, and stomach. In the present study, we have characterized the expression of GATA-4 and GATA-6 during development of another endodermal derivative, the mouse pancreas, using a combination of in situ hybridization and immunohistochemistry. Neither GATA-4 nor GATA-6 antigen was detected in E10.5 pancreatic epithelial buds expressing Pdx-1. By E15.5, GATA-4 mRNA and protein were evident in developing pancreatic acini, but not in ductal or endocrine cells of the pancreas; GATA-6 mRNA and protein were present in both endocrine and exocrine cell precursors. In the newborn and adult pancreas, GATA-4 protein was seen in acinar cells, while GATA-6 antigen was found mainly in islet beta-cells. The amphicrine pancreatic AR42J-B13 cell line was used to study the expression of GATA-4 and GATA-6 during the differentiation of these cells towards an endocrine phenotype. Endocrine differentiation was associated with marked increase in GATA-6 but not GATA-4 mRNA levels. We conclude that GATA-4 is a marker of exocrine pancreatic differentiation, whereas GATA-6 is a marker of endocrine pancreatic development.

Animals↗
PubMed2004

A syndrome with multiple malformations, mental retardation, and ACTH deficiency.

We report on a patient with severe pre- and post-natal growth retardation, moderate mental retardation, microcephaly, unusual face with marked micrognathia and cleft palate, minor skeletal abnormalities, atrioseptal defect, hypospadias, hearing loss, and secondary adrenal insufficiency due to isolated ACTH deficiency diagnosed at 7 years of age. Family history was negative. Adrenal insufficiency is an uncommon feature in multiple malformation syndromes and may thus serve as a diagnostic handle for recognizing other possible patients with a similar syndrome.

Abnormalities, Multiple↗
PubMed2004

Mechanisms of beta cell death during restricted and unrestricted enterovirus infection.

Coxsackie B virus (CVB-5) infections potentially trigger and accelerate pancreatic beta cell damage leading to type 1 diabetes. In vivo, all viruses face natural resistance mediated by various host factors which restrict the progression of infection. Thus, the aims of this study were to generate a tissue culture model of restricted coxsackie B virus infection in primary islet cells by preventing the production of viral progeny with a selective inhibitor of viral RNA replication and to investigate the mechanisms of virus-induced islet cell death during productive and restricted infective conditions. Cultured foetal porcine islet cells were infected effectively with the prototype strain of coxsackievirus B5 (CVB-5). Nuclear viability stainings and electron microscopy showed productive infection to result in dominantly necrotic cell death with additional slight induction of apoptosis during the 7 days of follow-up. The restricted conditions were created by addition of guanidine-hydrochloride (G-HCl) into culture medium. At 1 mM concentration, it significantly protected the infected cells from necrosis and thus maintained high viability. This was associated with increased significantly apoptosis. In perifusion analysis, the cellular ability to release insulin was reduced, although the metabolic integrity was preserved as shown by MTT-analysis and cellular ATP levels. These data show that restriction of CVB-5 replication with G-HCl protects islet cells against virus-induced necrosis. However, restriction of viral replication shifts the mechanism of cell death from necrosis toward apoptosis. A slowly progressing subclinical infection of islets could thus lead to increased beta-cell apoptosis.

Adenosine Triphosphate↗
PubMed2004

Mechanisms of coxsackievirus B5 mediated beta-cell death depend on the multiplicity of infection.

Coxsackievirus infections may trigger and accelerate pancreatic beta-cell death, leading to type I diabetes. Unrestricted coxsackievirus B5 replication in cultured beta-cells inoculated with high multiplicity leads to rapid lytic cell death. Evidence from other virus-host cell systems indicates that host cell responses to infection may depend on the multiplicity of infection (MOI). Thus, the aim of this study was to compare the mechanisms of beta-cell death during high versus low multiplicity of coxsackievirus B5 infection. Cultures of highly differentiated mouse insulinoma cells and primary adult human islets were infected with coxsackievirus B5 at multiplicities of >1,000 or <0.5 TCID50 per cell. The results of nuclear morphology and viability stainings, TUNEL staining and electrophoretic DNA fragmentation analysis showed high multiplicity infection to predominantly induce necrosis and transient apoptosis. In low multiplicity culture, however, necrosis was only moderately induced and apoptosis increased steadily with time. This was best demonstrated by a tenfold higher apoptosis/necrosis ratio than after high multiplicity inoculation. Expression of gamma-glutamyl cysteine synthetase increased in both infective cultures but the level of intracellular glutathione permanently depleted only at high multiplicity and recovered fully at low multiplicity. Thus, apoptosis represents an important mechanism of beta-cell death after low multiplicity of coxsackievirus B5 infection. This process is associated with maintenance of a physiological intracellular glutathione profile differing dramatically from the high multiplicity infection during which necrosis dominates and intracellular thiol balance deteriorates. These data suggest that the pattern and mechanisms of coxsackievirus B5 infection induced beta-cell death depend on the MOI.

Animals↗
PubMed2004

Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation.

Glucokinase (GK) is a glycolytic key enzyme that functions as a glucose sensor in the pancreatic beta-cell, where it governs glucose-stimulated insulin secretion (GSIS). Heterozygous inactivating mutations in the glucokinase gene (GCK) cause a mild form of diabetes (maturity-onset diabetes of the young [MODY]2), and activating mutations have been associated with a mild form of familial hyperinsulinemic hypoglycemia. We describe the first case of severe persistent hyperinsulinemic hypoglycemia due to a "de novo" mutation in GCK (Y214C). A baby girl presented with hypoglycemic seizures since the first postnatal day as well as with inappropriate hyperinsulinemia. Severe hypoglycemia persisted even after treatment with diazoxide and subtotal pancreatectomy, leading to irreversible brain damage. Pancreatic histology revealed abnormally large and hyperfunctional islets. The mutation is located in the putative allosteric activator domain of the protein. Functional studies of purified recombinant glutathionyl S-transferase fusion protein of GK-Y214C showed a sixfold increase in its affinity for glucose, a lowered cooperativity, and increased kcat. The relative activity index of GK-Y214C was 130, and the threshold for GSIS predicted by mathematical modeling was 0.8 mmol/l, compared with 5 mmol/l in the wild-type enzyme. In conclusion, we have identified a de novo GCK activating mutation that causes hyperinsulinemic hypoglycemia of exceptional severity. These findings demonstrate that the range of the clinical phenotype caused by GCK mutations varies from complete insulin deficiency to extreme hyperinsulinemia.

Amino Acid Substitution↗