Chris Burns

A Role for the Extracellular Calcium-Sensing Receptor in Cell-Cell Communication in Pancreatic Islets of Langerhans

Background: The extracellular calcium-sensing receptor (CaR) is expressed in many tissues that are not associated with Ca2+ homeostasis, including the endocrine cells in pancreatic islets of Langerhans. We have demonstrated previously that pharmacological activation of the CaR stimulates insulin secretion from islet β-cells and insulin-secreting MIN6 cells. Methods: In the present study we have investigated the effects of CaR activation on MIN6 cell proliferation and have used shRNA-mediated CaR knockdown to determine whether the CaR is involved in the regulation of insulin secretion via cell-cell communication. Results: CaR activation caused the phosphorylation and activation of the p42/44 MAPK signalling cascade, and this activation was prevented by the shRNA-induced down-regulation of CaR mRNA expression. CaR activation also resulted in increased proliferation of MIN6 cells, consistent with the known role of the p42/44 MAPK system in the regulation of β-cell proliferation. Down-regulation of CaR expression had no detectable effects on glucose-induced insulin secretion from MIN6 cells maintained as monolayers, but blocked the increases in insulin secretion that were observed when the cells were configured as three-dimensional islet-like structures (pseudoislets), consistent with a role for the CaR in cell-cell communication in pseudoislets. Conclusion: It is well established that islet function is dependent on communication between islet cells and the results of this study suggest that the CaR is required for β-cell to β-cell interactions within islet-like structures.

Preparation and validation of the first WHO international genetic reference panel for Fragile X syndrome

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by expansion of a trinucleotide (CGG)n repeat sequence in the 5′ untranslated region of the FMR1 gene, resulting in promoter hypermethylation and suppression of FMR1 transcription. Additionally, pre-mutation alleles in carrier males and females may result in Fragile X tremor ataxia syndrome and primary ovarian insufficiency, respectively. Fragile X is one of the most commonly requested molecular genetic tests worldwide. Quality assessment schemes have identified a wide disparity in allele sizing between laboratories. It is therefore important that clinical laboratories have access to characterized reference materials (RMs) to aid accurate allele sizing and diagnosis. With this in mind, a panel of genotyping RMs for Fragile X syndrome has been developed, which should be stable over many years and available to all diagnostic laboratories. Immortalized cell lines were produced by Epstein–Barr virus transformation of lymphocytes from consenting patients. Genomic DNA was extracted in bulk and RM aliquots were freeze-dried in glass ampoules. Twenty-one laboratories from seventeen countries participated in a collaborative study to assess their suitability. Participants evaluated the samples (blinded, in triplicate) in their routine methods alongside in-house and commercial controls. The panel of five genomic DNA samples was endorsed by the European Society of Human Genetics and approved as an International Standard by the Expert Committee on Biological Standardization at the World Health Organization.

The role of microRNAs in the pancreatic differentiation of pluripotent stem cells.

The generation of β-cells in vitro is an attractive option for cell therapy treatments for type 1 diabetes and also for the development of more accurate disease models. A number of studies have demonstrated that insulin-expressing cells can be generated by the in vitro differentiation of human pluripotent stem cells. However, to date, these differentiation protocols are often inefficient, time-consuming and highly variable. In many cases, this is a result of an incomplete understanding of the regulatory processes involved in the differentiation of human pluripotent stem cells. One such process is the control of gene expression by microRNAs (miRNAs). Given that miRNAs have the potential to influence cell fate, we present in this short review the evidence that a further understanding of the role of miRNAs in pancreatic development and function may be important in the on-going quest to generate insulin-secreting cells from pluripotent stem cells.

Changes in microRNA expression during differentiation of embryonic and induced pluripotent stem cells to definitive endoderm

Pluripotent stem cells, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have the potential to treat type 1 diabetes through cell replacement therapy. However, the protocols used to generate insulin-expressing cells in vitro frequently result in cells which have an immature phenotype and are functionally restricted. MicroRNAs (miRNAs) are now known to be important in cell fate specification, and a unique miRNA signature characterises pancreatic development at the definitive endoderm stage. Several studies have described differences in miRNA expression between ESCs and iPSCs. Here we have used microarray analysis both to identify miRNAs up- or down-regulated upon endoderm formation, and also miRNAs differentially expressed between ESCs and iPSCs. Several miRNAs fulfilling both these criteria were identified, suggesting that differences in the expression of these miRNAs may affect the ability of pluripotent stem cells to differentiate into definitive endoderm. The expression of these miRNAs was validated by qRT-PCR, and the relationship between one of these miRNAs, miR-151a-5p, and its predicted target gene, SOX17, was investigated by luciferase assay, and suggested an interaction between miR-151a-5p and this key transcription factor. In conclusion, these findings demonstrate a unique miRNA expression pattern for definitive endoderm derived from both embryonic and induced pluripotent stem cells.

Preparation, calibration and evaluation of the First International Standard for human C-peptide

Abstract Background: Measurement of C-peptide by immunoassay contributes to the diagnosis of a number of disorders related to β cell function. Stocks of the current international reference reagent (IRR) for C-peptide, used to calibrate these immunoassays, are exhausted, and this report summarises the international study to establish a replacement World Health Organization (WHO) international standard (IS) to maintain the availability of a globally available reference material and support efforts to standardise C-peptide assays. Methods: The study was conducted in three phases; phase I involved the assignment of a value to a primary calibrant in mass units by amino acid analysis and phase II applied this value to the calibration of a candidate standard, 13/146, by reversed phase high-performance liquid chromatography (RP-HPLC) assay. In phase III, the candidate standard was compared to the first IRR by current immunoassays to assess its suitability to serve as an IS. Results: Calibration of the candidate standard by RP-HPLC gave a final estimated content of 8.64 μg/ampoule with expanded uncertainty of 8.21–9.07 μg/ampoule (95% confidence; k=2.45). The candidate standard also appears sufficiently stable to serve as an IS, based on HPLC analysis of accelerated thermal degradation samples of 13/146, and was also shown to have appropriate immunological activity. A difference in bias approach was used to assess the commutability of 13/146 with human serum and urine samples. With the exception of two laboratories, the candidate standard demonstrated commutability with respect to the serum and urine samples included in this study. Conclusions: The candidate standard, 13/146, is suitable to serve as the First International Standard for human C-peptide, and it has been formally adopted by the Expert Committee on Biological Standardisation of the WHO.

Applications of cell-based bioassays measuring the induced expression of endogenous genes.

Cell-based bioassays are used to determine the biological activity of complex biotherapeutic products, to assign potency and to assure the quality and consistency of the manufacturing process. Clinically, these assays are used to assess bioactivity in patient samples, particularly for the detection of antidrug neutralizing antibodies. Owing to their versatility, cellular assays that measure endogenous gene expression by quantitative reverse transcription PCR offer a rapid and automatable alternative to assays measuring functional, late-stage responses. Notably, detection of immediate early gene expression represents a direct response of the cell to receptor ligation by the biotherapeutic. We review current developments in the use of this approach and demonstrate its application to the detection of receptor-binding autoantibodies using, as a case study, the detection of autoantibodies to the thyroid-stimulating hormone receptor.

Standardization of therapeutic, urinary gonadotrophins: An update on the use and availability of International Standards for Menotrophin

The potencies of therapeutic preparations of gonadotrophins of human, urinary origin, which comprise a heterogenous mix of isoforms with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) bioactivities, are standardized by WHO International Standards (IS). We report here, the evaluation, through an international collaborative study, of a candidate preparation, coded 10/286, to replace the 4th IS, 98/704, for human, urinary FSH and LH (Menotrophin) which has been used for many years for the potency assignment of therapeutic preparations using bioassays. The mean FSH and LH bioactivities of 10/286, determined by in vivo bioassays in terms of 98/704, were 183 IU per ampoule (95% confidence limits 165-202) and 177 IU per ampoule (95% confidence limits 159-197), respectively.