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Genetic heterogeneity of primary lesion and metastasis in small intestine neuroendocrine tumors
(2018)
Data on intratumoral heterogeneity of small intestine neuroendocrine tumors (SI-NETs) and related liver metastasis are limited. The aim of this study was to characterize genetic heterogeneity of 5 patients with SI-NETs. Therefore, formalin-fixed, paraffin-embedded tissue samples of primary and metastatic lesions as well as benign liver of five patients with synchronously metastasized, well differentiated SI-NETs were analyzed with whole exome sequencing. For one patient, chip based 850k whole DNA methylome analysis was performed of primary and metastatic tumor tissue as well as control tissue. Thereby, 156 single nucleotide variants (SNVs) in 150 genes were identified and amount of mutations per sample ranged from 9–34 (mean 22). The degree of common (0–94%) and private mutations per sample was strongly varying (6–100%). In all patients, copy number variations (CNV) were found and the degree of intratumoral heterogeneity of CNVs corresponded to SNV analysis. DNA methylation analysis of a patient without common SNVs revealed a large overlap of common methylated CpG sites. In conclusion, SI-NET primary and metastatic lesions show a highly varying degree of intratumoral heterogeneity. Driver events might not be detectable with exome analysis only, and further comprehensive studies including whole genome and epigenetic analyses are warranted.
Background: Definite diagnosis and therapeutic management of cholangiocarcinoma (CCA) remains a challenge. The aim of the current study was to investigate feasibility and potential impact on clinical management of targeted sequencing of intraductal biopsies.
Methods: Intraductal biopsies with suspicious findings from 16 patients with CCA in later clinical course were analyzed with targeted sequencing including tumor and control benign tissue (n = 55 samples). A CCA-specific sequencing panel containing 41 genes was designed and a dual strand targeted enrichment was applied.
Results: Sequencing was successfully performed for all samples. In total, 79 mutations were identified and a mean of 1.7 mutations per tumor sample (range 0–4) as well as 2.3 per biopsy (0–6) were detected and potentially therapeutically relevant genes were identified in 6/16 cases. In 14/18 (78%) biopsies with dysplasia or inconclusive findings at least one mutation was detected. The majority of mutations were found in both surgical specimen and biopsy (68%), while 28% were only present in biopsies in contrast to 4% being only present in the surgical tumor specimen.
Conclusion: Targeted sequencing from intraductal biopsies is feasible and potentially improves the diagnostic yield. A profound genetic heterogeneity in biliary dysplasia needs to be considered in clinical management and warrants further investigation.
Translational impact: The current study is the first to demonstrate the feasibility of sequencing of intraductal biopsies which holds the potential to impact diagnostic and therapeutical management of patients with biliary dysplasia and neoplasia.
Immune-modulating therapy is a promising therapy for patients with cholangiocarcinoma (CCA). Microsatellite instability (MSI) might be a favorable predictor for treatment response, but comprehensive data on the prevalence of MSI in CCA are missing. The aim of the current study was to determine the prevalence of MSI in a German tertiary care hospital. Formalin-fixed paraffin-embedded tissue samples, obtained in the study period from 2007 to 2015 from patients with CCA undergoing surgical resection with curative intention at Johann Wolfgang Goethe University hospital, were examined. All samples were investigated immunohistochemically for the presence of MSI (expression of MLH1, PMS2, MSH2, and MSH6) as well as by pentaplex polymerase chain reaction for five quasimonomorphic mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-22, and NR-24). In total, 102 patients were included, presenting intrahepatic (n = 35, 34.3%), perihilar (n = 42, 41.2%), and distal CCA (n = 25, 24.5%). In the immunohistochemical analysis, no loss of expression of DNA repair enzymes was observed. In the PCR-based analysis, one out of 102 patients was found to be MSI-high and one out of 102 was found to be MSI-low. Thus, MSI seems to appear rarely in CCA in Germany. This should be considered when planning immune-modulating therapy trials for patients with CCA.
Rationale: Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important regulators of inflammation. The exact impact of ROS/RNS on cutaneous delayed-type hypersensitivity reaction (DTHR) is controversial. The aim of our study was to identify the dominant sources of ROS/RNS during acute and chronic trinitrochlorobenzene (TNCB)-induced cutaneous DTHR in mice with differently impaired ROS/RNS production.
Methods: TNCB-sensitized wild-type, NADPH oxidase 2 (NOX2)- deficient (gp91phox-/-), myeloperoxidase-deficient (MPO-/-), and inducible nitric oxide synthase-deficient (iNOS-/-) mice were challenged with TNCB on the right ear once to elicit acute DTHR and repetitively up to five times to induce chronic DTHR. We measured ear swelling responses and noninvasively assessed ROS/RNS production in vivo by employing the chemiluminescence optical imaging (OI) probe L-012. Additionally, we conducted extensive ex vivo analyses of inflamed ears focusing on ROS/RNS production and the biochemical and morphological consequences.
Results: The in vivo L-012 OI of acute and chronic DTHR revealed completely abrogated ROS/RNS production in the ears of gp91phox-/- mice, up to 90 % decreased ROS/RNS production in the ears of MPO-/- mice and unaffected ROS/RNS production in the ears of iNOS-/- mice. The DHR flow cytometry analysis of leukocytes derived from the ears with acute DTHR confirmed our in vivo L-012 OI results. Nevertheless, we observed no significant differences in the ear swelling responses among all the experimental groups. The histopathological analysis of the ears of gp91phox-/- mice with acute DTHRs revealed slightly enhanced inflammation. In contrast, we observed a moderately reduced inflammatory immune response in the ears of gp91phox-/- mice with chronic DTHR, while the inflamed ears of MPO-/- mice exhibited the strongest inflammation. Analyses of lipid peroxidation, 8-hydroxy-2'deoxyguanosine levels, redox related metabolites and genomic expression of antioxidant proteins revealed similar oxidative stress in all experimental groups. Furthermore, inflamed ears of wild-type and gp91phox-/- mice displayed neutrophil extracellular trap (NET) formation exclusively in acute but not chronic DTHR.
Conclusions: MPO and NOX2 are the dominant sources of ROS/RNS in acute and chronic DTHR. Nevertheless, depletion of one primary source of ROS/RNS exhibited only marginal but conflicting impact on acute and chronic cutaneous DTHR. Thus, ROS/RNS are not a single entity, and each species has different properties at certain stages of the disease, resulting in different outcomes.
Alzheimer's disease-related mutations in the presenilin-1 gene (PS1) are leading to an elevated production of neurotoxic beta-amyloid 1-42 and may additionally enhance oxidative stress. Here, we provide in vivo evidence indicating that brains of transgenic mice expressing different human Alzheimer-linked PS1 mutations exhibit a reduced activity of two antioxidant enzymes. For this purpose, mice transgenic for human PS1 and for single and multiple PS1 mutations were generated. Mice with multiple PS1 mutations showed a significantly decreased activity of the antioxidant enzymes Cu/Zn superoxide dismutase and glutathione reductase already at an age of 3-4 months. As expected, this effect was less pronounced for the mice with a single PS1 mutation. By contrast, animals bearing normal human PS1 showed significantly elevated enzyme activities relative to non-transgenic littermate controls.
Many cases of early-onset inherited Alzheimer's disease (AD) are caused by mutations in the presenilin-1 (PS1) gene. Expression of PS1 mutations in cell culture systems and in primary neurons from transgenic mice increases their vulnerability to cell death. Interestingly, enhanced vulnerability to cell death has also been demonstrated for peripheral lymphocytes from AD patients. We now report that lymphocytes from PS1 mutant transgenic mice show a similar hypersensitivity to cell death as do peripheral cells from AD patients and several cell culture systems expressing PS1 mutations. The cell death-enhancing action of mutant PS1 was associated with increased production of reactive oxygen species and altered calcium regulation, but not with changes of mitochondrial cytochrome c. Our study further emphasizes the pathogenic role of mutant PS1 and may provide the fundamental basis for new efforts to close the gap between studies using neuronal cell lines transfected with mutant PS1, neurons from transgenic animals, and peripheral cells from AD patients. Copyright 2001 Academic Press.
The relevance of physiological immune aging is of great interest with respect to determining disorders with pathologic immune function in aging individuals. In recent years, the relevance of changes in peripheral lymphocytes in age-associated neurologic diseases has become more evident. Due to the lack of immunological studies, covering more than one event after mitogenic activation, we envisaged a new concept in the present study, aiming to investigate several events, starting from T cell receptor (TCR) ligation up to T cell proliferation. In addition, we addressed the question whether changes are present in the subsets (CD4, CD8) with aging. Phosphorylation of tyrosine residues declines with increasing age in CD4+ cells. Fewer levels of CD69 positive cells after 4 h mitogenic activation, altered expression of cytokines (IL2, IFN-gamma and TNF-alpha; 22 h) and lower proliferation (72 h) were determined in aging. Moreover, it could be shown that CD8+ lymphocytes react more effectively to mitogenic stimulation with reference to CD69 expression and proliferation in both age groups (<35 and >60 years old). These data indicate that T cell activation, mediated by TCR engagement, is significantly impaired in aging and both subsets are affected. However, bypassing the TCR does not fully restore T cell function, indicating that there are more mechanisms involved than impaired signal transduction through TCR only. The results will be discussed in relation to their relevance in neurodegenerative and psychiatric disorders.
Apoptosis seems to be involved in immunosenescence associated with aging. Moreover, in lymphocytes (PBL) of patients with Alzheimer's disease, an increased susceptibility to the apoptotic pathway has been described possibly due to impaired protection of oxidative stress. Accordingly, it seemed to be of particular interest to investigate the contribution of normal aging to the susceptibility from human lymphocytes to programmed cell death. We could show that PBL from elderly individuals (>60 years) accumulate apoptosing cells to a significant higher extent in spontaneous and activation-induced cell death compared to younger controls (<35 years). Treatment with the oxidative stressor 2-deoxy-D-ribose or with agonistic-CD95-antibody pronounced this effect even more implicating a higher sensitivity to reactive oxygen species and a higher functional CD95 expression, respectively. In addition, expression of the activation markers HLA-DR and CD95 was significantly increased in CD3+-cells of aged subjects, while expression of CD25 did not seem to be affected by age. Expression of Bcl-2 was increased in aging and correlated with the number of apoptotic cells.
Enhanced apoptosis and elevated levels of reactive oxygen species (ROS) play a major role in aging. In addition, several neurodegenerative diseases are associated with increased oxidative stress and apoptosis in neuronal tissue. Antioxidative treatment has neuro-protective effects. The aim of the present study was to evaluate changes of susceptibility to apoptotic cell death by oxidative stress in aging and its inhibition by the antioxidant Ginkgo biloba extract EGb761. We investigated basal and ROS-induced levels of apoptotic lymphocytes derived from the spleen in young (3 months) and old (24 months) mice. ROS were induced by 2-deoxy-D-ribose (dRib) that depletes the intracellular pool of reduced glutathione. Lymphocytes from aged mice accumulate apoptotic cells to a significantly higher extent under basal conditions compared to cells from young mice. Treatment with dRib enhanced this difference, implicating a higher sensitivity to ROS in aging. Apoptosis can be reduced in vitro by treatment with EGb761. In addition, mice were treated daily with 100mg/kg EGb761 per os over a period of two weeks. ROS-induced apoptosis was significantly reduced in the EGb761 group. Interestingly, this effect seemed to be more pronounced in old mice.
As major sources of reactive oxygen species (ROS), mitochondrial structures are exposed to high concentrations of ROS and may therefore be particularly susceptible to oxidative damage. Mitochondrial damage could play a pivotal role in the cell death decision. A decrease in mitochondrial energy charge and redox state, loss of transmembrane potential (depolarization), mitochondrial respiratory chain impairment, and release of substances such as calcium and cytochrome c all contribute to apoptosis. These mitochondrial abnormalities may constitute a part of the spectrum of chronic oxidative stress in Alzheimer's disease. Accumulation of amyloid beta (Abeta) in form of senile plaques is also thought to play a central role in the pathogenesis of Alzheimer's disease mediated by oxidative stress. In addition, increasing evidence shows that Abeta generates free radicals in vitro, which mediate the toxicity of this peptide. In our study, PC12 cells were used to examine the protective features of EGb 761(definition see editorial) on mitochondria stressed with hydrogen peroxide and antimycin, an inhibitor of complex III. In addition, we investigated the efficacy of EGb 761 in Abeta-induced MTT reduction in PC12 cells. Moreover, we examined the effects of EGb 761 on ROS levels and ROS-induced apoptosis in lymphocytes from aged mice after in vivo administration. Here, we will report that EGb 761 was able to protect mitochondria from the attack of hydrogen peroxide, antimycin and Abeta. Furthermore, EGb 761 reduced ROS levels and ROS-induced apoptosis in lymphocytes from aged mice treated orally with EGb 761 for 2 weeks. Our data further emphasize neuroprotective properties of EGb 761, such as protection against Abeta-toxicity, and antiapoptotic properties, which are probably due to its preventive effects on mitochondria.
Introduction: Reactive oxygen species (ROS) have been implicated in neurodegeneration and seem to be involved in the physiology and pathophysiology of several diseases, including normal aging and Alzheimer’s disease (AD). Enhanced ROS production in aging or AD is not restricted to the brain, but can also been seen in several peripheral tissues. The objective of the present study was to evaluate whether the mechanisms involved in the generation of oxidative stress in normal senescence and Alzheimer’s disease are identical or not. Methods: We analysed intracellular basal levels of ROS in lymphocytes from AD patients and healthy young and aged not-demented subjects as well as ROS levels following stimulation with d-ribose and staurosporine in all three groups. ROS levels were measured by flow cytometry using the intracellular fluorescence dye dihydrorhodamine123 (DHR123). Results: Our study shows that AD lymphocytes have increased basal levels of ROS, low susceptibility to ROS stimulation by 2-deoxy-D-ribose (dRib) and an increased response to staurosporine when compared with age-matched controls. Discussion: The data suggest that the defect(s) responsible for enhanced ROS production in AD may involve different or additional biological pathways than those involved in enhanced ROS generation during aging.
The identification of specific genetic (presenilin-1 [PS1] and amyloid precursor protein [APP] mutations) and environmental factors responsible for Alzheimer's disease (AD) has revealed evidence for a shared pathway of neuronal death. Moreover, AD-specific cell defects may be observed in many other nonneuronal cells (e.g., lymphocytes). Thus, lymphocytes may serve as a cellular system in which to study risk factors of sporadic, as well as genetic AD in vivo. The aim of our present study was to clarify whether lymphocytes bearing genetic or sporadic risk factors of AD share an increased susceptibility to cell death. Additionally we examined whether a cell typespecific vulnerability pattern was present and how normal aging, the main risk factor of sporadic AD, contributes to changes in susceptibility to cell death. Here, we report that lymphocytes affected by sporadic or genetic APP and PS1 AD risk factors share an increased vulnerability to cell death and exhibit a similar cell type-specific pattern, given that enhanced vulnerability was most strongly developed in the CD4+ T-cell subtype. In this paradigm, sporadic risk factors revealed the highest impact on cell type-specific sensitivity of CD4+ T cells to apoptosis. In contrast, normal aging results in an increased susceptibility to apoptosis of both, CD4+ and CD8+ T cells.
Poster presentation: Here we investigated the role of the amyloid precursor protein (APP) in regulation of Ca2+ store depletion-induced neural cell death. Ca2+ store depletion from the endoplasmic reticulum (ER) was induced by the SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase) inhibitor thapsigargin which led to a rapid induction of the unfolded protein response (UPR) and a delayed activation of executioner caspases in the cultures. Overexpression of APP potently enhanced cytosolic Ca2+ levels and cell death after ER Ca2+ store depletion in comparison to vector-transfected controls. GeneChipR and RT-PCR analysis revealed that the expression of classical UPR chaperone genes was not altered by overexpression of APP.Interestingly, the induction of the ER stress-responsive pro-apoptotic transcription factor CHOP was significantly upregulated in APP-overexpressing cells in comparison to vectortransfected controls. Chelation of intracellular Ca2+ with BAPTA-AM revealed that enhanced CHOP expression after store depletion occured in a Ca2+-dependent manner in APPoverexpressing cells. Prevention of CHOP induction by BAPTA-AM and by RNA interference was also able to abrogate the potentiating effect of APP on thapsigargin-induced apoptosis. Application of the store-operated channel (SOC)-inhibitors SK F96365 and 2-APB downmodulated APP-triggered potentiation of cytosolic Ca2+ levels and apoptosis after treatment with thapsigargin. Our data demonstrate that APP-mediated regulation of ER Ca2+ homeostasis significantly modulates Ca2+ store depletion-induced cell death in a SOC- and CHOP-dependent manner, but independent of the UPR.
Mapping cortical brain asymmetry in 17,141 healthy individuals worldwide via the ENIGMA Consortium
(2017)
The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.
Cdc2-like kinases (CLKs) represent a family of serine-threonine kinases involved in the regulation of splicing by phosphorylation of SR-proteins and other splicing factors. Although compounds acting against CLKs have been described, only a few show selectivity against dual-specificity tyrosine phosphorylation regulated-kinases (DYRKs). We here report a novel CLK inhibitor family based on a 6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one core scaffold. Within the series, 3-(3-chlorophenyl)-6,7-dihydropyrrolo[3,4-g]indol-8(1H)-one (KuWal151) was identified as inhibitor of CLK1, CLK2 and CLK4 with a high selectivity margin towards DYRK kinases. The compound displayed a potent antiproliferative activity in an array of cultured cancer cell lines. The X-ray structure analyses of three members of the new compound class co-crystallized with CLK proteins corroborated a molecular binding mode predicted by docking studies.
Particle production as a function of charged-particle flattenicity in pp collisions at √s = 13 TeV
(2024)
This paper reports the first measurement of the transverse momentum (pT) spectra of primary charged pions, kaons, (anti)protons, and unidentified particles as a function of the charged-particle flattenicity in pp collisions at s√=13 TeV. Flattenicity is a novel event shape observable that is measured in the pseudorapidity intervals covered by the V0 detector, 2.8<η<5.1 and −3.7<η<−1.7. According to QCD-inspired phenomenological models, it shows sensitivity to multiparton interactions and is less affected by biases towards larger pT due to local multiplicity fluctuations in the V0 acceptance than multiplicity. The analysis is performed in minimum-bias (MB) as well as in high-multiplicity events up to pT=20 GeV/c. The event selection requires at least one charged particle produced in the pseudorapidity interval |η|<1. The measured pT distributions, average pT, kaon-to-pion and proton-to-pion particle ratios, presented in this paper, are compared to model calculations using PYTHIA 8 based on color strings and EPOS LHC. The modification of the pT-spectral shapes in low-flattenicity events that have large event activity with respect to those measured in MB events develops a pronounced peak at intermediate pT (2<pT<8 GeV/c), and approaches the vicinity of unity at higher pT. The results are qualitatively described by PYTHIA, and they show different behavior than those measured as a function of charged-particle multiplicity based on the V0M estimator.
Measurement of beauty production via non-prompt charm hadrons in p-Pb collisions at √sNN = 5.02 TeV
(2024)
The production cross sections of D0, D+, and Λ+c hadrons originating from beauty-hadron decays (i.e. non-prompt) were measured for the first time at midrapidity in proton−lead (p−Pb) collisions at the center-of-mass energy per nucleon pair of √sNN=5.02 TeV. Nuclear modification factors (RpPb) of non-prompt D0, D+, and Λ+c are calculated as a function of the transverse momentum (pT) to investigate the modification of the momentum spectra measured in p−Pb collisions with respect to those measured in proton−proton (pp) collisions at the same energy. The RpPb measurements are compatible with unity and with the measurements in the prompt charm sector, and do not show a significant pT dependence. The pT-integrated cross sections and pT-integrated RpPb of non-prompt D0 and D+ mesons are also computed by extrapolating the visible cross sections down to pT = 0. The non-prompt D-meson RpPb integrated over pT is compatible with unity and with model calculations implementing modification of the parton distribution functions of nucleons bound in nuclei with respect to free nucleons. The non-prompt Λ+c/D0 and D+/D0 production ratios are computed to investigate hadronisation mechanisms of beauty quarks into mesons and baryons. The measured ratios as a function of pT display a similar trend to that measured for charm hadrons in the same collision system.
The production yields of antideuterons and antiprotons are measured in pp collisions at a center-of-mass energy of √s=13 TeV, as a function of transverse momentum (pT) and rapidity (y), for the first time up to |y|=0.7. The measured spectra are used to study the pT and rapidity dependence of the coalescence parameter B2, which quantifies the coalescence probability of antideuterons. The pT and rapidity dependence of the obtained B2 is extrapolated for pT>1.7 GeV/c and |y|>0.7 using the phenomenological antideuteron production model implemented in PYTHIA 8.3 as well as a baryon coalescence afterburner model based on EPOS 3. Such measurements are of interest to the astrophysics community, since they can be used for the calculation of the flux of antinuclei from cosmic rays, in combination with coalescence models.