Effects of different extraction buffers on peanut protein detectability and lateral flow device (LFD) performance.

The accidental uptake of peanuts can cause severe health reactions in allergic individuals. Reliable determination of traces of peanuts in food products is required to support correct labelling and therefore minimise consumers' risk. The immunoanalytical detectability of potentially allergenic peanut proteins is dependent on previous heat treatment, the extraction capacity of the applied buffer and the specificity of the antibody. In this study a lateral flow device (LFD) for the detection of peanut protein was developed and the capacity of 30 different buffers to extract proteins from mildly and strongly roasted peanut samples as well as their influence on the test strip performance were investigated. Most of the tested buffers showed good extraction capacity for putative Ara h 1 from mildly roasted peanuts. Protein extraction from dark-roasted samples required denaturing additives, which were proven to be incompatible with LFD performance. High-pH buffers increased the protein yield but inhibited signal generation on the test strip. Overall, the best results were achieved using neutral phosphate buffers but equal detectability of differently altered proteins due to food processing cannot be assured yet for immunoanalytical methods.

Adult human beta-cell neogenesis?

Prospects for inducing endogenous beta-cell regeneration in the pancreas, one of the most attractive approaches to reverse type 1 and type 2 diabetes, have gained substantially from recent evidence that cells in the adult pancreas exhibit more plasticity than previously recognized. There are two major pathways to beta-cell regeneration, beta-cell replication and beta-cell neogenesis. Substantial evidence for a role for both processes exists in different models. While beta-cell replication clearly occurs during development and early in life, the potential for replication appears to decline substantially with age. In contrast, we have demonstrated that the exocrine compartment of the adult human pancreas contains a facultative stem cell that can differentiate into beta-cells under specific circumstances. We have favoured the idea that, similar to models described in liver regeneration, beta-cell mass can be increased either by neogenesis or replication, depending on the intensity of different stimuli or stressors. Understanding the nature of endocrine stem/progenitor cells and the mechanism by which external stimuli mobilize them to exhibit endocrine differentiation is central for success in therapeutic approaches to induce meaningful endogenous beta-cell neogenesis.

NeuroD1 in the endocrine pancreas: localization and dual function as an activator and repressor.

The basic helix-loop-helix transcription factor NeuroD1 regulates cell fate in the nervous system but previously has not been considered to function similarly in the endocrine pancreas due to its reported expression in all islet cell types in the newborn mouse. Because we found that NeuroD1 potently represses somatostatin expression in vitro, its pattern of expression was examined in both strains of mice in which lacZ has been introduced into the NeuroD1 locus by homologous recombination. Analysis of adult transgenic mice revealed that NeuroD1 is predominantly expressed in beta-cells and either absent or expressed below the limit of lacZ detection in mature alpha-, delta-, or PP cells. Consistent with a previous report, NeuroD1 colocalizes with glucagon as well as insulin in immature islets of the newborn mouse. However, no colocalization of NeuroD1with somatostatin was detected in the newborn. In vitro, ectopic expression of NeuroD1 in TRM-6/PDX-1, a human pancreatic delta-cell line, resulted in potent repression of somatostatin concomitant with induction of the beta-cell hormones insulin and islet amyloid polypeptide. Additionally, NeuroD1 induced expression of Nkx2.2, a transcription factor expressed in beta- but not delta-cells. Transfection studies using insulin and somatostatin promoters confirm the ability of NeuroD1 to act as both a transcriptional repressor and activator in the same cell, suggesting a more complex role for NeuroD1 in the establishment and/or maintenance of mature endocrine cells than has been recognized previously.

Beta-cell differentiation from a human pancreatic cell line in vitro and in vivo.

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human beta-cells, inducibly transformed pancreatic beta-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of beta-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional beta-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human beta-cells is a major step toward developing a cell transplantation therapy for diabetes.

PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line.

Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.

Sustained proliferation of PDX-1+ cells derived from human islets.

Ex vivo expansion of human beta-cells is an important step toward the development of cell-based insulin delivery systems in type 1 diabetes. Here, we report that human pancreatic endocrine cells can be expanded through 15 cell doublings in vitro for an estimated total 30,000-fold increase in cell number. We believe that the cells resulting from these cultures are of beta-cell origin, since they uniformly express the transcription factor PDX-1 (STF-1, IDX-1, IPF-1), which is initially seen only in cells positive for insulin and negative for the ductal cell marker cytokeratin (CK)-19. To rule out the possibility that PDX-1 expression might be induced by the culture conditions used here, cells from isolated human pancreatic ducts were cultured under the same conditions as the islet cells. Cells in these cultures expressed CK-19 but not PDX-1. Although the expanded beta-cells continued to express PDX-1, insulin expression was lost over time. Whether reexpression of islet-specific genes in vitro is essential for successful cell transplantation remains to be determined.

Interleukin-10 inhibits expression of both interferon alpha- and interferon gamma- induced genes by suppressing tyrosine phosphorylation of STAT1.

Interleukin-10 (IL-10) helps maintain polarized T-helper cells in a T-helper lymphocyte 2 (Th2) phenotype. Part of this process involves the prevention of the development of Th1 cells, which are a primary source of interferon gamma (IFNgamma), a potent activator of monocytes and an inhibitor of Th2 proliferation. Because monocytes and macrophages are important mediators of Th1-type responses, such as delayed-type hypersensitivity, we sought to determine if IL-10 could directly mediate inhibition of IFNgamma- and IFNalpha-induced gene expression in these cells. Highly purified monocytes were incubated with IL-10 for 60 to 90 minutes before the addition of IFNgamma or IFNalpha. IL-10 preincubation resulted in the inhibition of gene expression for several IFN-induced genes, such as IP-10, ISG54, and intercellular adhesion molecule-1. The reduction in gene expression resulted from the ability of IL-10 to suppress IFN-induced assembly of signal transducer and activator of transcription (STAT) factors to specific promoter motifs on IFNalpha- and IFNgamma-inducible genes. This was accomplished by preventing the IFN-induced tyrosine phosphorylation of STAT1, a component of both IFNalpha- and IFNgamma-induced DNA binding complexes. Therefore, IL-10 can directly inhibit STAT-dependent early response gene expression induced by both IFNalpha and IFNgamma in monocytes by suppressing the tyrosine phosphorylation of STAT1. This may occur through the ability of IL-10 to induce expression of the gene, suppressor of cytokine signaling 3 (SOCS3).

Isolation and characterization of a cell line from the epithelial cells of the human fetal pancreas.

Pancreatic cell lines are useful for basic studies of pancreatic biology and for possible application to cell transplantation therapies for diabetes. A retroviral vector expressing simian virus 40 (SV40) T antigen and H-rasval12 was used to infect a monolayer culture of epithelial cells from an 18-wk human fetal pancreas. Infected cells gave rise to a clonal epithelial cell line, designated TRM-1. This cell line expresses epithelial markers as well as gult2 and small amounts of insulin and glucagon. TRM-1 is the first cell line to be generated from the human fetal pancreas and also the first cell line derived directly from the fetal pancreas of any species. The approach that we have used to develop TRM-1 should be applicable to isolating cell lines from other stages of human pancreatic development.

E2A gene products are not required for insulin gene expression.

Transcripts for E2A gene products, ubiquitous basic helix-loop-helix transactivating proteins, are expressed at high levels in the pancreatic epithelium. E2A proteins have been shown to bind the cognate E box sequence (CANNTG) of the insulin promoter/enhancer. E2A gene products dimerize with cell-specific basic helix-loop-helix proteins and synergize with the homeodomain transcription factor, PDX-1, in insulin gene transactivation. PDX-1 is also required for normal pancreatic development in mice. We investigated whether pancreatic development and insulin production could occur in the absence of E2A gene products by studying mice with a null mutation for the gene. E2A(-/-) mice demonstrated normal formation of pancreatic endocrine and exocrine tissue in histochemical sections as well as positive and distinct immunostaining for insulin and glucagon in islet tissue, signifying development of mature beta- and alpha-cells. Moreover, E2A(-/-) mice displayed no significant difference in blood glucose levels or pancreatic insulin content compared with wild-type littermates. These data show that although E2A gene products probably play an important role in insulin gene expression, pancreatic development and insulin production can proceed in their absence.

Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis.