Clemens Hufnagl

Effect of the AMP-Kinase Modulators AICAR, Metformin and Compound C on Insulin Secretion of INS-1E Rat Insulinoma Cells under Standard Cell Culture Conditions

Background/Aims: The function of β-cells is regulated by nutrient uptake and metabolism. The cells′ metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of KATP channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to KATP channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and β-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat β-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, KATP channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose). Methods: Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry. Results: We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 µM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of KATP currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 µM) led to a depolarization of Vm, which was not due to an inhibition of the basal- or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Cai). AICAR had no additive blocking effect on KATP currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration <3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity. Conclusion: We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms.

A modified scoring of the NCCN-IPI is more accurate in the elderly and is improved by albumin and β2-microglobulin

The International Prognostic Index (IPI) has been used for decades in diffuse large B‐cell lymphoma (DLBCL). A retrospective cancer registry analysis in North America showed significantly improved results when an enhanced IPI, the National Comprehensive Cancer Network (NCCN)‐IPI was applied. This novel score puts more weight on age and high levels of lactate dehydrogenase (LDH). Nevertheless, it remains unclear if these results can be extrapolated to the general population. This retrospective bi‐centre analysis included 499 unselected DLBCL patients who were treated with rituximab and anthracycline‐based chemoimmunotherapy between 2004 and 2013. In our cohort, the NCCN‐IPI was more accurate in identifying patients at low or high risk, despite older age, and more patients with increased LDH. Nevertheless, a modified scoring of the risk factors was required to more accurately identify elderly patients with a very favourable diagnosis, suggesting an impaired value of the original NCCN‐IPI in the elderly. Serum β2‐microglobulin and albumin were retained as independent prognostic factors for survival in a multivariate analysis. Our data confirm, for the first time, the superior prognostic power of the NCCN‐IPI in an unselected, middle‐European cohort. We furthermore propose a modified NCCN‐IPI for more accurate prognostication in the elderly. Albumin and β2‐microglobulin levels are likely to add significant information to the NCCN‐IPI.

Effect of combined Low-Dose Radon- and Hyperthermia Treatment (LDRnHT) of patients with ankylosing spondylitis on serum levels of cytokines and bone metabolism markers: a pilot study

Ankylosing Spondylitis (AS) is a rheumatic disease and almost 50% of the affected patients suffer from osteoporosis. The ratio of Receptor Activator of Nuclear Factor κB Ligand (RANKL) and Osteoprotegerin (OPG) has become an important marker to assess the status of systemic bone metabolism and has been shown to be dysregulated in AS patients. Combined Low-Dose Radon- and Hyperthermia Therapy (LDRnHT) causes pain reduction in patients with AS and induces the cytokine transforming growth factor-β1 (TGF-β1). TGF-β1 acts as an anti-inflammatory cytokine and influences the OPG/RANK/RANKL system. We therefore performed a study on 33 AS patients to investigate the effect of LDRnHT on serum levels of bone metabolism markers and cytokines involved in chronic inflammatory disorders. It is shown that TGF-β1, TNF-α, IL-6, OPG and RANKL levels significantly increased after LDRnHT and that the ratio of OPG over RANKL is significantly elevated.

miR-16-5p Is a Stably-Expressed Housekeeping MicroRNA in Breast Cancer Tissues from Primary Tumors and from Metastatic Sites

For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)® microarrays from Agilent® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.

The phytostilbene resveratrol induces apoptosis in INS-1E rat insulinoma cells.

We investigated the effect of resveratrol on proliferation and induction of apoptosis of INS-1E rat insulinoma cells by cell counting, crystal violet staining, flow cytometry and immunoblotting. Resveratrol treatment of INS-1E cells at concentrations ≥50 μM resulted in a dose-dependent inhibition of cell proliferation, accumulation of the cells in the S and G0/G1 phase and a significant increase of the percentage of apoptotic cells. This was paralleled by an increase of cell granularity, apoptotic volume decrease (AVD), exposure of phosphatidylserine at the outer leaflet of the plasma membrane, an increase of the 7-AAD signal and caspase activation. The AMP-kinase (AMPK) inhibitor compound C (10 μM) significantly inhibited cell proliferation and induced caspase activation within 48 hours but this effect was not modified by resveratrol suggesting that AMPK is not a major target involved in mediating the proapoptotic effect of resveratrol in INS-1E cells. Immunoblotting revealed a significant inhibition of Akt (PKB) phosphorylation by 100 μM resveratrol within 1 hour. Addition of insulin (10 μM) to the culture medium strongly enhanced basal Akt phosphorylation. This enhancement was significantly attenuated by 50 and 100 μM resveratrol. We conclude that the antiproliferative/proapoptotic effect of resveratrol on INS-1E cells is due to negative interference with Akt signaling and most likely disruption of auto/paracrine insulin signaling.

Clonal evolution in relapsed and refractory diffuse large B-cell lymphoma is characterized by high dynamics of subclones

Little information is available about the role of certain mutations for clonal evolution and the clinical outcome during relapse in diffuse large B-cell lymphoma (DLBCL). Therefore, we analyzed formalin-fixed-paraffin-embedded tumor samples from first diagnosis, relapsed or refractory disease from 28 patients using next-generation sequencing of the exons of 104 coding genes. Non-synonymous mutations were present in 74 of the 104 genes tested. Primary tumor samples showed a median of 8 non-synonymous mutations (range: 0-24) with the used gene set. Lower numbers of non-synonymous mutations in the primary tumor were associated with a better median OS compared with higher numbers (28 versus 15 months, p=0.031). We observed three patterns of clonal evolution during relapse of disease: large global change, subclonal selection and no or minimal change possibly suggesting preprogrammed resistance. We conclude that targeted re-sequencing is a feasible and informative approach to characterize the molecular pattern of relapse and it creates novel insights into the role of dynamics of individual genes.

Was bringt Glaukom-Screening?

SummaryGlaucoma is a disease that satisfies many of the essential criteria for a screening program. The increasing prevalence of the disease and the rapidly aging population are indicators that advanced visual defects caused by glaucoma as well as the resulting costs will increase significantly in the near future. Early diagnosis makes early rather inexpensive treatment possible. Early treatment slows progression and prevents blindness and unnecessary suffering.ZusammenfassungGlaukom ist eine Erkrankung, die die wichtigsten Kriterien eines Screeningprogrammes erfüllt. Die zunehmende Häufigkeit der Erkrankung und die rasch älter werdende Bevölkerung lassen vermuten, dass sowohl fortgeschrittene, durch Glaukom bedingte Sehbeeinträchtigungen als auch die daraus entstehenden Kosten in der nahen Zukunft rasch ansteigen werden. Da durch Frühdiagnose eine rechtzeitig einsetzende kostengünstige Therapie möglich ist und damit die Erkrankung in ihrer Progression positiv beeinflusst wird, kann durch Glaukom-Screening Blindheit und unnötiges Leid vermieden werden.

Beeinflussen verschiedene Hornhaut-Kulturmethoden das Transplantatüberleben?

ZusammenfassungVerschiedene Kultivierungsmethoden, Spenderalter, Hornhautdicke, Endothelzellzahl und andere Parameter werden für das Transplantatüberleben unterschiedlich diskutiert. An unserer Klinik werden Hornhäute von Spendern einerseits zu verschiedenen Post-mortem-Zeiten (von Einzeloder Multiorganspendern) und andererseits in verschiedenen Kultivierungsmedien (Organkultur versus Kurzzeitmedium Optisol) bis zur Transplantation aufbewahrt. Wir untersuchten in einer retrospektiven Studie, ob sich dadurch für die Hornhautempfänger Unterschiede im Transplantatüberleben nach perforierender Keratoplastik ergeben. 315 transplantierte Hornhäute wurden nach 2 Jahren evaluiert. Die verschiedenen Kulturmethoden, Anzahl der Endothelzellen, Hornhautdicke, Spenderalter und der jeweilige Transplantatdurchmesser wurden berücksichtigt. Zusätzlich wurde zwischen Multioder Einzelorganspendern unterschieden.Prima vista zeigte sich ein besseres Transplantatüberleben zugunsten der Optisol-konservierten Spenderorgane. Da jedoch die Hornhäute unterschiedlich „verteilt“ wurden, war es notwendig, die zugrundeliegenden Erkrankungen und die untersuchten Parameter einzeln aufzuschlüsseln und die jeweiligen Gruppen zu vergleichen. Dadurch wurde klar ersichtlich, dass weder die unterschiedlichen Post-mortem-Zeiten (bis zu 24 Stunden) noch die verschiedenen Kulturmethoden bzw. die Dauer der Kultur zu signifikanten Unterschieden im Transplantatüberleben führten. Ebensowenig hatten das Spenderalter und die Dicke der Hornhaut vor Kultur einen signifikanten Einfluss auf das Transplantatüberleben. Auch kein Unterschied zwischen Corneae von Multioder Einzelorganspendern war feststellbar. Der bedeutendste prognostische Faktor war die zugrunde liegende Primärerkrankung.SummaryDifferent culture methods, donor age, cornea thickness, endothelial cell density and other parameters are differently discussed for graft survival. We use donor corneas with different post mortem times (from single- and multiorgan-donors) and various culture methods for grafts. In a retrospective study we investigated the influence of the mentioned parameters in a follow-up of graft survival after penetrating keratoplasty. 315 corneas were transplanted and followed up to two years. We investigated if different culture methods, endothelial cell density, cornea thickness, donor age and graft diameter had influence on graft survival. Additionally we could discriminate between grafts from single-or multiorgan-donors.We first found a better graft survival with grafts from Optisol. But, as the grafts were not randomly distributed, we further had to divide our patients in various graft-rejection risk groups. Thereafter, we could not find any significant influences on graft survival depending on post mortem times (up to 24 hours) or different culture methods or storage times. Also donor age and cornea thickness had no significant influence on graft survival. Further, grafts from multi-or single organ donors did not differ in their long-term survival. The most important prognostic factor was the underlying disease which led to transplantation.

Clonal evolution and heterogeneity in metastatic head and neck cancer—An analysis of the Austrian Study Group of Medical Tumour Therapy study group

BACKGROUND Tumour heterogeneity and clonal evolution within a cancer patient are deemed responsible for relapse in malignancies and present challenges to the principles of targeted therapy, for which treatment modality is often decided based on the molecular pathology of the primary tumour. Nevertheless, the clonal architecture in distant relapse of head and neck cancer is fairly unknown. PATIENTS AND METHODS For this project, we analysed a cohort of 386 patients within the Austrian Registry of head and neck cancer. We identified 26 patients with material from the primary tumour, the distant metastasis after curative first-line treatment and a germline sample for analysis of clonal evolution. After pathological analyses, these samples were analysed using a targeted massively parallel sequencing (MPS) panel of 257 genes known to be recurrently mutated in head and neck cancer plus a genome-wide SNP-set. RESULTS Despite histological diagnosis of distant metastasis, no corresponding mutation in the supposed metastases was found in two of 23 (8.6%) evaluable patients suggesting a primary tumour of the lung instead of a distant metastasis of head and neck cancer. We observed a branched pattern of evolution in 31.6% of the analysed patients. This pattern was associated with a shorter time to distant metastasis, compared with a pattern of punctuated evolution. Structural genomic changes over time were also present in 7 of 12 (60%) evaluable patients with metachronous metastases. CONCLUSION Targeted MPS demonstrated substantial heterogeneity at the time of diagnosis and a complex pattern of evolution during disease progression in head and neck cancer. Copy number analyses revealed additional changes that were not detected by mutational analyses. Mutational and structural changes contribute to tumour heterogeneity at diagnosis and progression.

Treatment of aggressive B-cell lymphoma in elderly patients: influence of single nucleotide polymorphisms affecting pharmacodynamics of chemotherapeutics

Abstract Clinical and/or biological risk factors are needed to identify elderly patients with aggressive B-cell lymphoma able to receive full-dose R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) treatment. We present a retrospective analysis of 83 patients ≥ 75 years of age (range: 75–97) who were diagnosed with aggressive B cell lymphoma between 2004 and 2011 in our clinic. R-CHOP-like therapy was administered in 82% of these patients resulting in a median overall survival of 54 months. A median cumulative dose of 226 mg/m2 doxorubicin and a median of six cycles were applied in these patients. Two genotypes of the CBR3 and MLH1 genes affecting the metabolism of cytostatics identified a subgroup with a favorable prognosis (median overall survival not reached vs. 30 months, p = 0.01). A treatment strategy aiming at full-dose R-CHOP was feasible and resulted in an encouraging treatment outcome in patients ≥ 75 years. Pharmacogenetic parameters, if independently validated, may be helpful in elderly patients.