100,000 Genomes Pilot on Rare-Disease Diagnosis in Health Care - Preliminary Report.

BACKGROUND: The U.K. 100,000 Genomes Project is in the process of investigating the role of genome sequencing in patients with undiagnosed rare diseases after usual care and the alignment of this research with health care implementation in the U.K. National Health Service. Other parts of this project focus on patients with cancer and infection. METHODS: We conducted a pilot study involving 4660 participants from 2183 families, among whom 161 disorders covering a broad spectrum of rare diseases were present. We collected data on clinical features with the use of Human Phenotype Ontology terms, undertook genome sequencing, applied automated variant prioritization on the basis of applied virtual gene panels and phenotypes, and identified novel pathogenic variants through research analysis. RESULTS: Diagnostic yields varied among family structures and were highest in family trios (both parents and a proband) and families with larger pedigrees. Diagnostic yields were much higher for disorders likely to have a monogenic cause (35%) than for disorders likely to have a complex cause (11%). Diagnostic yields for intellectual disability, hearing disorders, and vision disorders ranged from 40 to 55%. We made genetic diagnoses in 25% of the probands. A total of 14% of the diagnoses were made by means of the combination of research and automated approaches, which was critical for cases in which we found etiologic noncoding, structural, and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohortwide burden testing across 57,000 genomes enabled the discovery of three new disease genes and 19 new associations. Of the genetic diagnoses that we made, 25% had immediate ramifications for clinical decision making for the patients or their relatives. CONCLUSIONS: Our pilot study of genome sequencing in a national health care system showed an increase in diagnostic yield across a range of rare diseases. (Funded by the National Institute for Health Research and others.).

Whole-genome sequencing of patients with rare diseases in a national health system.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

Faecal microbiota transplantation as a treatment for inflammatory bowel disease: a national survey of adult and paediatric gastroenterologists in the UK.

BACKGROUND: Interest in the use of faecal microbiota transplantation (FMT) in inflammatory bowel disease (IBD) has increased following outcomes in patients with Clostridioides difficile infection (CDI). While research exploring clinician awareness and attitude towards the use of FMT in CDI has been carried out, data for IBD are currently lacking. OBJECTIVE: To assess the perceptions of gastroenterologists and current practice relating to FMT as a treatment for IBD in the UK. DESIGN: A web-based survey (Snap Survey software) was distributed through the British Society of Gastroenterology (BSG) and British Society of Paediatric Gastroenterology, Hepatology and Nutrition e-newsletters, and at the BSG Conference in June 2017. RESULTS: 61 respondents completed the survey including presubspecialty trainees, gastroenterology specialists, associate specialists and consultants. Most (95%; n=58) respondents stated that they had heard of FMT being used as a treatment for IBD prior to participating in the survey. Based on current evidence, 34% (n=21) of respondents would consider using FMT in patients with IBD, 26% (n=16) would not and 39% (n=24) were undecided. When asked to rank routes of delivery in terms of preference, nasogastric tube was the least preferred route (39%; n=24) and oral capsule was the most preferred route (34%; n=21). CONCLUSIONS: A clear majority of UK gastroenterologists recognise FMT as a potential treatment for IBD; however, uptake is limited. A proportion of clinicians would consider FMT in IBD and the majority would consider entering patients into clinical trials. Future work should explore the utility and efficacy of oral FMT capsules in IBD.

Elevated Levels of Alpha Cells Emanating from the Pancreatic Ducts of a Patient with a Low BMI and Chronic Pancreatitis.

Chronic pancreatitis (CP) is an inflammatory disease that causes progressive damage to the pancreatic parenchyma with irreversible morphological changes and fibrotic replacement of the gland. The risk factors associated with developing CP have been described as toxic (e.g., alcohol and tobacco); idiopathic (e.g., unknown); genetic, autoimmune, recurrent acute pancreatitis, and obstructive (the TIGAR-O system). Upon histological screening of the pancreata from a cohort of CP patients who had undergone pancreatectomy for the treatment of intractable pain in Leicester, UK, one sample showed a striking change in the morphological balance toward an endocrine phenotype, most notably there was evidence of substantial α cell genesis enveloping entire cross sections of ductal epithelium and the presence of α cells within the ductal lumens. This patient had previously undergone a partial pancreatectomy, had severe sclerosing CP, an exceptionally low body mass index (15.2), and diabetes at the time the pancreas was removed, and although these factors have been shown to induce tissue remodeling, such high levels of α cells was an unusual finding within our series of patients. Due to the fact that α cells have been shown to be the first endocrine cell type that emerges during islet neogenesis, future research profiling the factors that caused such marked α cell genesis may prove useful in the field of islet transplantation.

Performance Differences Among Skilled Soccer Players of Different Playing Positions During Vertical Jumping and Landing.

Harry, JR, Barker, LA, James, CR, and Dufek, JS. Performance differences among skilled soccer players of different playing positions during vertical jumping and landing. J Strength Cond Res 32(2): 304-312, 2018-Both jumping and landing performance of skilled soccer players is diminished when task demands are increased. However, it is unclear if performance changes are specific to players of certain playing positions. Therefore, we assessed jumping and landing performance among skilled soccer players of different playing positions. Twenty-five National Collegiate Athletic Association (NCAA) Division 1 male soccer players (179.5 ± 7.8 cm, 75.5 ± 7.1 kg, 19.7 ± 1.2 years) performed maximum effort vertical jump landings (VJLs), whereas vertical ground reaction force (vGRF) data were obtained. Participants were stratified into goalkeeping (GK), defensive (DEF), midfield (MID), and attacking (ATT) group according to their primary playing position. One-way analyses of variance (α = 0.05) and effect sizes (ESs; large ≥ 0.80) were used to compare differences among groups. The jumping phase variables evaluated were jump height, unloading and amortization vGRF magnitudes, eccentric rate of force development, and the reactive strength index. Landing phase variables included the peak vGRF magnitude, vGRF loading rate, vGRF attenuation rate, and landing time. No statistically significant differences were detected for any jumping or landing variable (p ≥ 0.05). However, a number of large magnitude differences were detected during landing after ES calculations. Specifically, greater peak vGRF magnitudes were detected in DEF vs. both MID (ES = 1.08) and ATT (ES = 0.93), a greater vGRF loading rate occurred in DEF vs. MID (ES = 0.93), and a greater vGRF attenuation rate occurred in DEF vs. both MID (ES = 1.00) and AT (ES = 0.80). It is concluded that highly skilled soccer players possess position-specific abilities with respect to the landing phase of VJL. Skilled soccer players might experience enhanced training outcomes after VJL training regimens tailored to the specific demands of their primary playing position.

The Human Phenotype Ontology in 2017.

Deep phenotyping has been defined as the precise and comprehensive analysis of phenotypic abnormalities in which the individual components of the phenotype are observed and described. The three components of the Human Phenotype Ontology (HPO; www.human-phenotype-ontology.org) project are the phenotype vocabulary, disease-phenotype annotations and the algorithms that operate on these. These components are being used for computational deep phenotyping and precision medicine as well as integration of clinical data into translational research. The HPO is being increasingly adopted as a standard for phenotypic abnormalities by diverse groups such as international rare disease organizations, registries, clinical labs, biomedical resources, and clinical software tools and will thereby contribute toward nascent efforts at global data exchange for identifying disease etiologies. This update article reviews the progress of the HPO project since the debut Nucleic Acids Research database article in 2014, including specific areas of expansion such as common (complex) disease, new algorithms for phenotype driven genomic discovery and diagnostics, integration of cross-species mapping efforts with the Mammalian Phenotype Ontology, an improved quality control pipeline, and the addition of patient-friendly terminology.

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic ß-Cells.

Saturated stearic acid (SA) induces apoptosis in the human pancreatic ß-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic ß-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.

Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor.

Enteropathogenic Escherichia coli (EPEC) are a major cause of infant diarrhoea in developing countries and a significant public health issue in industrialized countries. Currently there are no simple tests available for the diagnosis of EPEC. Serology of O-antigens is widely used routinely in many laboratories throughout the world, even though it has been known for many years to be an unreliable indicator of EPEC virulence. We have developed a simple, low-cost immunodiagnostic test based on the EspA filament, an essential virulence factor of EPEC and the related enterohaemorrhagic E. coli (EHEC). Using recombinant proteins of the five major variants of EspA as immunogens, we raised a panel of three monoclonal antibodies in mice that detects all variants of the native target in bacterial cultures. The antibodies proved suitable for application in sandwich-type assays, including ELISA and lateral flow immunoassays (LFI). Prototypes for both assays were specific for EPEC and EHEC strains when tested against a panel of control micro-organisms. We have also developed a simple, affordable culture medium, A/E medium, which optimizes expression of EspA allowing improved sensitivity of detection compared with standard Dulbecco's modified Eagle's medium. Together these reagents form the basis of robust, informative tests for EPEC for use especially in developing countries but also for routine screening in any clinical laboratory.

Mitomycin or cisplatin chemoradiation with or without maintenance chemotherapy for treatment of squamous-cell carcinoma of the anus (ACT II): a randomised, phase 3, open-label, 2 × 2 factorial trial.

BACKGROUND: Chemoradiation became the standard of care for anal cancer after the ACT I trial. However, only two-thirds of patients achieved local control, with 5-year survival of 50%; therefore, better treatments are needed. We investigated whether replacing mitomycin with cisplatin in chemoradiation improves response, and whether maintenance chemotherapy after chemoradiation improves survival. METHODS: In this 2 × 2 factorial trial, we enrolled patients with histologically confirmed squamous-cell carcinoma of the anus without metastatic disease from 59 centres in the UK. Patients were randomly assigned to one of four groups, to receive either mitomycin (12 mg/m(2) on day 1) or cisplatin (60 mg/m(2) on days 1 and 29), with fluorouracil (1000 mg/m(2) per day on days 1-4 and 29-32) and radiotherapy (50.4 Gy in 28 daily fractions); with or without two courses of maintenance chemotherapy (fluorouracil and cisplatin at weeks 11 and 14). The random allocation was generated by computer and patients assigned by telephone. Randomisation was done by minimisation and stratified by tumour site, T and N stage, sex, age, and renal function. Neither patients nor investigators were masked to assignment. Primary endpoints were complete response at 26 weeks and acute toxic effects (for chemoradiation), and progression-free survival (for maintenance). The primary analyses were done by intention to treat. This study is registered at controlled-trials.com, number 26715889. FINDINGS: We enrolled 940 patients: 472 were assigned to mitomycin, of whom 246 were assigned to no maintenance, 226 to maintenance; 468 were assigned to cisplatin, of whom 246 were assigned to no maintenance, 222 to maintenance. Median follow-up was 5.1 years (IQR 3.9-6.9). 391 of 432 (90.5%) patients in the mitomycin group versus 386 of 431 (89.6%) in the cisplatin group had a complete response at 26 weeks (difference -0.9%, 95% CI -4.9 to 3.1; p=0.64). Overall, toxic effects were similar in each group (334/472 [71%] for mitomycin vs 337/468 [72%] for cisplatin). The most common grade 3-4 toxic effects were skin (228/472 [48%] vs 222/468 [47%]), pain (122/472 [26%] vs 135/468 [29%]), haematological (124/472 [26%] vs 73/468 [16%]), and gastrointestinal (75/472 [16%] vs 85/468 [18%]). 3-year progression-free survival was 74% (95% CI 69-77; maintenance) versus 73% (95% CI 68-77; no maintenance; hazard ratio 0.95, 95% CI 0.75-1.21; p=0.70). INTERPRETATION: The results of our trial--the largest in anal cancer to date--show that fluorouracil and mitomycin with 50.4 Gy radiotherapy in 28 daily fractions should remain standard practice in the UK. FUNDING: Cancer Research UK.

Caspase-2 and JNK activated by saturated fatty acids are not involved in apoptosis induction but modulate ER stress in human pancreatic ß-cells.

BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic ß-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic ß-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic ß-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.

The potential benefit of non-purified islets preparations for islet transplantation.

Since the advent of islet transplantation, there has been a significant emphasis on the importance of islet purity despite an inevitable associated loss of islet mass during the purification process. One of the key elements of the 'Edmonton Protocol' for islet transplantation published in 2000 was an emphasis on the need for sequential transplants of highly purified islets (averaging 24% beta cell purity) and the close correlation between the numbers of islets transplanted and the success of the procedure. However, the emphasis on islet purity may warrant further consideration as auto transplantation of non-purified islets currently provides the most successful insulin independence rates within the field of islet transplantation. While the role of auto and allo immunity could contribute to the differences in the success rates it is clear that within the clinical setting, significant acinar and ductal contamination is well tolerated. However, one could go further and hypothesize that extra-insular tissue including acinar tissue, ductal tissue, peri-pancreatic lymph nodes and vascular tissue actually confer an advantage to islet survival/function and may even contribute to the insulin secreting capacity of the graft post transplant. As such this review will assess the influence of extra-insular pancreatic tissue on the results of islet transplantation based on published evidence and will also explore the possibility that non-islet pancreatic cells are capable of differentiating into a beta cell phenotype in vivo contributing to an ongoing regeneration of endocrine mass during the period following transplantation.

Inhibitory effect of unsaturated fatty acids on saturated fatty acid-induced apoptosis in human pancreatic ß-cells: activation of caspases and ER stress induction.

AIMS: In this study we have tested the effect of unsaturated fatty acids on the proapoptotic effects of saturated fatty acids in the human pancreatic ß-cells NES2Y. RESULTS: We found that unsaturated palmitoleic and oleic acid at a concentration of 0.2 mM and higher are able to completely inhibit the proapoptotic effect of their counterpart saturated palmitic and stearic acid at a concentration of 1 mM. Apoptosis induced by stearic acid was associated with significant activation of caspase-6, -7, -9, -2 and -8, but not with significant activation of caspase-3. The activation of caspases was blocked by coincubation with oleic acid. Stearic acid treatment was not associated with a significant change in mitochondrial membrane potential, reactive oxygen species level and with cytochrome c release from mitochondria. Furthermore, stearic acid treatment was not associated with changes in p21(WAF1/CIP1), PIDD, Fas receptor and Fas ligand expression. However, we detected endoplasmic reticulum (ER) stress markers, i. e. a significant upregulation of BiP and CHOP expression as well as XBP1 mRNA splicing. These changes were inhibited by coincubation with oleic acid. CONCLUSION: Presented data indicate that oleic acid inhibits apoptosis induction by stearic acid in NES2Y cells upstream of caspase activation and ER stress induction. It does not involve an interference with the mitochondrial pathway of apoptosis induction, with p53 activation and PIDD expression as well as with Fas receptor and Fas ligand expression.

"Mind the gap"--the impact of variations in the duration of the treatment gap and overall treatment time in the first UK Anal Cancer Trial (ACT I).

PURPOSE: The United Kingdom Coordinating Committee on Cancer Research anal cancer trial demonstrated the benefit of combined modality treatment (CMT) using radiotherapy (RT), infusional 5-fluorouracil, and mitomycin C over RT alone. The present study retrospectively examines the impact of the recommended 6-week treatment gap and local RT boost on long-term outcome. METHODS AND MATERIALS: A total of 577 patients were randomly assigned RT alone or CMT. After a 6-week gap responders received a boost using either additional external beam radiotherapy (EBRT) (15 Gy) or iridium-192 implant (25 Gy). The effect of boost, the gap between initial treatment (RT alone or CMT) and boost (Tgap), and overall treatment time (OTT) were examined for their impact on outcome. RESULTS: Among the 490 good responders, 436 (89%) patients received a boost after initial treatment. For boosted patients, the risk of anal cancer death decreased by 38% (hazard ratio [HR]: 0.62, 99% CI 0.35-1.12; p=0.04), but there was no evidence this was mediated via a reduction in locoregional failure (LRF) (HR: 0.90, 99% CI 0.48-1.68; p=0.66). The difference in Tgap was only 1.4 days longer for EBRT boost, compared with implant (p=0.51). OTT was longer by 6.1 days for EBRT (p=0.006). Tgap and OTT were not associated with LRF. Radionecrosis was reported in 8% of boosted, compared with 0% in unboosted patients (p=0.03). CONCLUSIONS: These results question the benefit of a radiotherapy boost after a 6-week gap. The higher doses of a boost may contribute more to an increased risk of late morbidity, rather than local control.

Business intelligence and capacity planning: web-based solutions.

Income (activity) and expenditure (costs) form the basis of a modern hospital's 'business intelligence'. However, clinical engagement in business intelligence is patchy. This article describes the principles of business intelligence and outlines some recent developments using web-based applications.

Splenic rupture as a presenting feature of endocarditis.

We describe the first case of infective endocarditis presenting with spontaneous splenic rupture. Our patient, a known intravenous drug user presented with hypovolaemic shock secondary to splenic rupture. The patient was resuscitated and underwent an emergency splenectomy. Subsequent clinical examination revealed a systolic murmur and a diagnosis of mitral valve infective endocarditis was made after echocardiography. Splenic tissue, blood cultures and mitral valve tissue all cultured Enterococcus faecalis. The patient had a successful mitral valve replacement and was discharged home after 44 days. To our knowledge this is the first reported case of enterococcal endocarditis presenting with splenic rupture. This case highlights the need to consider endocarditis in spontaneous splenic rupture particularly in those patients in a high risk group, such as IV drug users, especially if they lack a clear history of trauma.

Characterisation of the NES2Y cell line and its use in the production of human glucose-responsive insulin producing (hGRIP) cell lines by cell-cell fusion.

Diabetes mellitus is a debilitating disease and alternative methods of treatment are a priority if the short-term and long term sequelae are to be avoided.  Here the authors manipulate NES2Y cells, which have the potential to be used as 'fusion partners' to produce human insulin-producing glucose-responsive hybrids.  The fusion experiments were carried out using polyethylene glycol (PEG) and electroporation.  Human insulin production of the resulting hybrids (in response to glucose) was measured using ELISA. Our results showed that it is possible to engineer human glucose-responsive insulin-producing (hGRIPs) hybrid cells by the manipulation of two different cell types. The resulting hybrids continuously grow in culture and are insulin-secreting and glucose-responsive for a period of time.  Immortalised cells with the characteristics of human beta cells could provide an important resource for experimental studies in Type 1 diabetes, such as an improved understanding of the fundamental mechanisms of glucose-responsive insulin processing and secretion, transplantation and drug screening programs.

Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit.

Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.

Comparison of the effect of individual saturated and unsaturated fatty acids on cell growth and death induction in the human pancreatic beta-cell line NES2Y.

We tested the effects of various types of fatty acids, differing in the degree of saturation and in the cis/trans configuration of the double bond, on the growth and viability of NES2Y cells (a human pancreatic beta-cell line). We found that during a 48-hour incubation period, saturated fatty acids, i.e. palmitic and stearic acids, at a physiologically relevant concentration of 1 mM and higher concentrations induced death of the beta-cells while their counterpart unsaturated fatty acids, i.e. palmitoleic and oleic acids, did not induce cell death at concentrations up to 3 mM. We also found that unsaturated elaidic acid with a trans double bond exerted significant inhibition of growth of the beta-cells at a concentration approximately ten times lower, i.e. 0.1 mM vs. 1 mM, than counterpart oleic acid with a cis double bond. This is the first direct evidence that a trans unsaturated fatty acid is significantly more effective in inhibiting beta-cell growth than a counterpart cis unsaturated fatty acid. Furthermore, we newly demonstrated that beta-cell death induced by saturated fatty acids is related to significant increase of caspase-2 activity (2 to 5-fold increase) but not to caspase-3 activation. The growth-inhibiting effect of saturated fatty acids at concentrations lower than death-inducing concentrations correlates with certain increase of caspase-2 activity.

Immunohistochemical evidence that culture in the high aspect rotating vessel can up-regulate hormone expression in growth dedifferentiated PHHI-derived islet cells.

Islet cells derived from patients with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) have the ability to grow readily in simple culture media. However, as with primary islets and cell lines, they lose hormone expression upon growth. In this study, we have investigated the role of three-dimensional cell-to-cell contact in the reinitiation of hormone expression in growth dedifferentiated PHHI-derived cells. Two main methods of cell aggregation were studied; the promotion of pseudoislets through petri dish culture and the creation of cell aggregates in the microgravity environment of the high aspect ratio vessel (HARV). Immunohistochemical analysis and ELISA assay showed that petri dish culture did not re-establish endocrine expression in any of the five cultures tested. However, through HARV technology, we have demonstrated that it is possible to reactivate insulin, glucagon, somatostatin, and GAD expression in PHHI-derived cells that had previously stopped expressing these markers. These results indicate that the unique environment of the HARV can be conducive to the upregulation of endocrine expression of islet-derived cells and optimization of culture conditions may prove useful in the sphere of beta cell proliferation.