The assessment of human islets for transplantation and the effect of the immunosuppressant rapamycin on MIN-6 cells, rat and human islets

Human islet transplantation is currently successfully establishing a role in the management of certain patients with type 1 diabetes. For islet transplantation to be successful there must be an adequate supply of high quality islets, which are damaged neither by host immunological defences nor the immunosuppressants used. In an attempt to characterise human islet quality several tests were developed. The static stimulation test assesses beta-cell function and responsiveness to glucose by measuring insulin release. To assess the degree of exocrine contamination of the islet preparation, the amylase content of both the whole pancreas and islet preparation were measured. Measuring the insulin content of both the whole pancreas and islet preparation allowed us to quantify the increase in insulin concentration as a consequence of the islet isolation procedure. The mean exocrine contamination (EC) was 2.5 +/- 0.7%, mean insulin enrichment (IE fold increase) 180 +/- 37 and mean static stimulation index (SSI) was 1.47 +/- 0.08 suggesting that the islet preparations have low levels of exocrine contamination, have significant insulin enrichment and release insulin in response to glucose. Thus the islet isolation procedure is reasonably successful at separating the endocrine from the exocrine pancreas. The curative potential of each islet material was then determined by transplanting representative islet samples to non-obese diabetic mice with severe combined immunodeficiency disease (NOD-SCID) as the biological endpoint in a standardised system. In the first two weeks following transplant, the degree of EC of the islet preparation may have some role in predicting the in-vivo function of islets, with those preparations of highest EC taking longer to restore normoglycaemia than the other groups. However, beyond 14 days post-transplant, EC is of no value in predicting the in-vivo function of islets. Similarly the degree of IE and the SSI have no value in predicting the in-vivo function of islets in this model. Thus, none of the biochemical indices (EC, IE nor SSI) are able to predict the in-vivo effectiveness or function of transplanted islets in the NOD-SCID mouse model. Non-heart beating donors (NHBDs) are generally not deemed suitable for whole organ pancreas donation but could provide a significant additional source of pancreata for islet transplantation if it was demonstrated that NHBD islets functioned no differently from islets isolated from heart-beating donors (HBDs). The recovery of islets from NHBDs was comparable to that of control HBD. In-vitro assessment of NHBD islet function revealed function equivalent to those isolated from HBD, and NHBD islets transplanted to NOD-SCID mice efficiently reversed diabetes. A single donor transplant from a NHBD resulted in a state of stable insulin independence in a type 1 diabetic recipient. Thus, normally functioning islets can be isolated successfully from NHBD pancreata, suggesting that NHBDs may provide an untapped source of pancreatic tissue for preparation of isolated islets for clinical transplantation. Rapamycin (sirolimus) is a macrolide fungicide with immunosuppressant properties that is used in human islet transplantation. Little is known about the effects of rapamycin on islets and MIN-6 cells (mouse clonal insulin-producing cells that are used experimentally as a beta-cell model). Rapamycin had a dose-dependent, time-dependent, glucose-independent deleterious effect on MIN-6 cell viability. At day 1, using the MTT method (this mitochondrial succinate dehydrogenase activity assay is used as an indirect measure of cell viability), 0.01 nM rapamycin reduced cell viability to 83 +/- 6% of control (p<0.05). Using the calcein AM method (a fluorescent marker of live cells), at day 2, 10 nM rapamycin caused a reduction in cell viability to 73 +/- 5% of control (p<0.001). Furthermore, 10 and 100 nM rapamycin caused apoptosis in MIN-6 cells as assessed by the TUNEL assay (apoptotic nicked DNA is fluorescinated and detected at the single cell level by flow cytometry). Compared to control, there was a 3.1 +/- 0.6 fold increase (p<0.01) in apoptosis in MIN-6 cells treated with 10 nM rapamycin. A supra- therapeutic rapamycin concentration of 100 nM significantly impaired glucose- and carbachol-stimulated insulin secretion of rat islets and had a deleterious effect on the viability of rat and human islets. Thus, currently there is no evidence that therapeutic concentrations of rapamycin have any in-vitro deleterious effect on islets

RUNX-mediated growth arrest and senescence are attenuated by diverse mechanisms in cells expressing RUNX1 fusion oncoproteins

RUNX gene over-expression inhibits growth of primary cells but transforms cells with tumor suppressor defects, consistent with reported associations with tumor progression. In contrast, chromosomal translocations involving RUNX1 are detectable in utero, suggesting an initiating role in leukemias. How do cells expressing RUNX1 fusion oncoproteins evade RUNX-mediated growth suppression? Previous studies showed that the TEL-RUNX1 fusion from t(12;21) B-ALLs is unable to induce senescence-like growth arrest (SLGA) in primary fibroblasts while potent activity is displayed by the RUNX1-ETO fusion found in t(8;21) AMLs. We now show that SLGA potential is suppressed in TEL-RUNX1 but reactivated by deletion of the TEL HLH domain or mutation of a key residue (K99R). Attenuation of SLGA activity is also a feature of RUNX1-ETO9a, a minor product of t(8;21) translocations with increased leukemogenicity. Finally, while RUNX1-ETO induces SLGA it also drives a potent senescence-associated secretory phenotype (SASP), and promotes the immortalisation of rare cells that escape SLGA. Moreover, the RUNX1-ETO SASP is not strictly linked to growth arrest as it is largely suppressed by RUNX1 and partially activated by RUNX1-ETO9a. These findings underline the heterogeneous nature of premature senescence and the multiple mechanisms by which this failsafe process is subverted in cells expressing RUNX1 oncoproteins

Clarifying the relationship between metformin, acute kidney injury and lactic acidosis

No abstract available

Addiction to Runx1 is partially attenuated by loss of p53 in the Eμ-Myc lymphoma model

The Runx genes function as dominant oncogenes that collaborate potently with Myc or loss of p53 to induce lymphoma when over-expressed. Here we examined the requirement for basal Runx1 activity for tumor maintenance in the Eµ-Myc model of Burkitt’s lymphoma. While normal Runx1fl/fl lymphoid cells permit mono-allelic deletion, primary Eµ-Myc lymphomas showed selection for retention of both alleles and attempts to enforce deletion in vivo led to compensatory expansion of p53null blasts retaining Runx1. Surprisingly, Runx1 could be excised completely from established Eµ- Myc lymphoma cell lines in vitro without obvious effects on cell phenotype. Established lines lacked functional p53, and were sensitive to death induced by introduction of a temperature-sensitive p53 (Val135) allele. Transcriptome analysis of Runx1-deleted cells revealed a gene signature associated with lymphoid proliferation, survival and differentiation, and included strong de-repression of recombination-activating (Rag) genes, an observation that was mirrored in a panel of human acute leukemias where RUNX1 and RAG1,2 mRNA expression were negatively correlated. Notably, despite their continued growth and tumorigenic potential, Runx1null lymphoma cells displayed impaired proliferation and markedly increased sensitivity to DNA damage and dexamethasone-induced apoptosis, validating Runx1 function as a potential therapeutic target in Myc-driven lymphomas regardless of their p53 status

The Relationship between AKI and CKD in Patients with Type 2 Diabetes:An Observational Cohort Study

Background There are few observational studies evaluating the risk of AKI in people with type 2 diabetes, and even fewer simultaneously investigating AKI and CKD in this population. This limits understanding of the interplay between AKI and CKD in people with type 2 diabetes compared with the nondiabetic population. Methods In this retrospective, cohort study of participants with or without type 2 diabetes, we used electronic healthcare records to evaluate rates of AKI and various statistical methods to determine their relationship to CKD status and further renal function decline. Results We followed the cohort of 16,700 participants (9417 with type 2 diabetes and 7283 controls without diabetes) for a median of 8.2 years. Those with diabetes were more likely than controls to develop AKI (48.6% versus 17.2%, respectively) and have preexisting CKD or CKD that developed during follow-up (46.3% versus 17.2%, respectively). In the absence of CKD, the AKI rate among people with diabetes was nearly five times that of controls (121.5 versus 24.6 per 1000 person-years). Among participants with CKD, AKI rate in people with diabetes was more than twice that of controls (384.8 versus 180.0 per 1000 person-years after CKD diagnostic date, and 109.3 versus 47.4 per 1000 person-years before CKD onset in those developing CKD after recruitment). Decline in eGFR slope before AKI episodes was steeper in people with diabetes versus controls. After AKI episodes, decline in eGFR slope became steeper in people without diabetes, but not among those with diabetes and preexisting CKD. Conclusions Patients with diabetes have significantly higher rates of AKI compared with patients without diabetes, and this remains true for individuals with preexisting CKD.on behalf of the BEAt-DKD Consortiu

A MAPK/c-Jun-mediated switch regulates the initial adaptive and cell death responses to mitochondrial damage in a neuronal cell model

Parkinson’s disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor cJun can act in either a pro- or anti-apoptotic manner, depending on the level ofstress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategie

The ugrizYJHK luminosity distributions and densities from the combined MGC, SDSS and UKIDSS LAS datasets

We combine data from the MGC, SDSS and UKIDSS LAS surveys to produce ugrizYJHK luminosity functions and densities from within a common, low redshift volume (z<0.1, ~71,000 h_1^-3 Mpc^3 for L* systems) with 100 per cent spectroscopic completeness. In the optical the fitted Schechter functions are comparable in shape to those previously reported values but with higher normalisations (typically 0, 30, 20, 15, 5 per cent higher phi*-values in u, g, r, i, z respectively over those reported by the SDSS team). We attribute these to differences in the redshift ranges probed, incompleteness, and adopted normalisation methods. In the NIR we find significantly different Schechter function parameters (mainly in the M* values) to those previously reported and attribute this to the improvement in the quality of the imaging data over previous studies. This is the first homogeneous measurement of the extragalactic luminosity density which fully samples both the optical and near-IR regimes. Unlike previous compilations that have noted a discontinuity between the optical and near-IR regimes our homogeneous dataset shows a smooth cosmic spectral energy distribution (CSED). After correcting for dust attenuation we compare our CSED to the expected values based on recent constraints on the cosmic star-formation history and the initial mass function.Comment: 17 pages, 13 figures, Accepted in MNRAS: 2010 January 18; in original form 2009 August 1

Human ASPM participates in spindle organisation, spindle orientation and cytokinesis

Background Mutations in the Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC). Results We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells. Conclusions These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype

A prospective evaluation of thiamine and magnesium status in relation to clinicopathological characteristics and 1-year mortality in patients with Alcohol Withdrawal Syndrome

Background: Alcohol withdrawal syndrome (AWS) is routinely treated with B-vitamins. However, the relationship between thiamine status and outcome is rarely examined. The aim of the present study was to examine the relationship between thiamine and magnesium status in patients with AWS. Methods: Patients (n = 127) presenting to the Emergency Department with AWS were recruited to a prospective observational study. Blood samples were drawn to measure whole blood thiamine diphosphate (TDP) and serum magnesium concentrations. Routine biochemistry and haematology assays were also conducted. The Glasgow Modified Alcohol Withdrawal Score (GMAWS) measured severity of AWS. Seizure history and current medications were also recorded. Results: The majority of patients (99%) had whole blood TDP concentration within/above the reference interval (275–675 ng/gHb) and had been prescribed thiamine (70%). In contrast, the majority of patients (60%) had low serum magnesium concentrations (&lt; 0.75 mmol/L) and had not been prescribed magnesium (93%). The majority of patients (66%) had plasma lactate concentrations above 2.0 mmol/L. At 1 year, 13 patients with AWS had died giving a mortality rate of 11%. Male gender (p &lt; 0.05), BMI &lt; 20 kg/m2 (p &lt; 0.01), GMAWS max ≥ 4 (p &lt; 0.05), elevated plasma lactate (p &lt; 0.01), low albumin (p &lt; 0.05) and elevated serum CRP (p &lt; 0.05) were associated with greater 1-year mortality. Also, low serum magnesium at time of recruitment to study and low serum magnesium at next admission were associated with higher 1-year mortality rates, (84% and 100% respectively; both p &lt; 0.05). Conclusion: The prevalence of low circulating thiamine concentrations were rare and it was regularly prescribed in patients with AWS. In contrast, low serum magnesium concentrations were common and not prescribed. Low serum magnesium was associated more severe AWS and increased 1-year mortality