Heat shock protein amplification improves cerebellar myelination in the Npc1nih mouse model.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare prematurely fatal lysosomal lipid storage disease with limited therapeutic options. The prominent neuropathological hallmarks include hypomyelination and cerebellar atrophy. We previously demonstrated the efficacy of recombinant human heat shock protein 70 (rhHSP70) in preclinical models of the disease. It reduced glycosphingolipid levels in the central nervous system (CNS), improving cerebellar myelination and improved behavioural phenotypes in Npc1nih (Npc1-/-) mice. Furthermore, treatment with arimoclomol, a well-characterised HSP amplifier, attenuated lysosomal storage in NPC patient fibroblasts and improved neurological symptoms in Npc1-/- mice. Taken together, these findings prompted the investigation of the effects of HSP amplification on CNS myelination. METHODS: We administered bimoclomol daily or rhHSP70 6 times per week to Npc1-/- (BALB/cNctr-Npc1m1N/J, also named Npc1nih) mice by intraperitoneal injection from P7 through P34 to investigate the impact on CNS myelination. The Src-kinase inhibitor saracatinib was administered with/without bimoclomol twice daily to explore the contribution of Fyn kinase to bimoclomol's effects. FINDINGS: Treatment with either bimoclomol or rhHSP70 improved myelination and increased the numbers of mature oligodendrocytes (OLs) as well as the ratio of active-to-inactive forms of phosphorylated Fyn kinase in the cerebellum of Npc1-/- mice. Additionally, treatment with bimoclomol preserved cerebellar weight, an effect that was abrogated when co-administered with saracatinib, an inhibitor of Fyn kinase. Bimoclomol-treated mice also exhibited increased numbers of immature OLs within the cortex. INTERPRETATION: These data increase our understanding of the mechanisms by which HSP70 regulates myelination and provide further support for the clinical development of HSP-amplifying therapies in the treatment of NPC. FUNDING: Funding for this study was provided by Orphazyme A/S (Copenhagen, Denmark) and a Pathfinder Award from The Wellcome Trust.

Galactosyl- and glucosylsphingosine induce lysosomal membrane permeabilization and cell death in cancer cells.

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments.

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87-118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Efficacy and safety of arimoclomol in Niemann-Pick disease type C: Results from a double-blind, randomised, placebo-controlled, multinational phase 2/3 trial of a novel treatment.

Niemann-Pick disease type C (NPC) is a rare, genetic, progressive neurodegenerative disorder with high unmet medical need. We investigated the safety and efficacy of arimoclomol, which amplifies the heat shock response to target NPC protein misfolding and improve lysosomal function, in patients with NPC. In a 12-month, prospective, randomised, double-blind, placebo-controlled, phase 2/3 trial (ClinicalTrials.gov identifier: NCT02612129), patients (2-18 years) were randomised 2:1 to arimoclomol:placebo, stratified by miglustat use. Routine clinical care was maintained. Arimoclomol was administered orally three times daily. The primary endpoint was change in 5-domain NPC Clinical Severity Scale (NPCCSS) score from baseline to 12 months. Fifty patients enrolled; 42 completed. At month 12, the mean progression from baseline in the 5-domain NPCCSS was 0.76 with arimoclomol vs 2.15 with placebo. A statistically significant treatment difference in favour of arimoclomol of -1.40 (95% confidence interval: -2.76, -0.03; P = .046) was observed, corresponding to a 65% reduction in annual disease progression. In the prespecified subgroup of patients receiving miglustat as routine care, arimoclomol resulted in stabilisation of disease severity over 12 months with a treatment difference of -2.06 in favour of arimoclomol (P = .006). Adverse events occurred in 30/34 patients (88.2%) receiving arimoclomol and 12/16 (75.0%) receiving placebo. Fewer patients had serious adverse events with arimoclomol (5/34, 14.7%) vs placebo (5/16, 31.3%). Treatment-related serious adverse events (n = 2) included urticaria and angioedema. Arimoclomol provided a significant and clinically meaningful treatment effect in NPC and was well tolerated.

Clinical disease progression and biomarkers in Niemann-Pick disease type C: a prospective cohort study.

BACKGROUND: Niemann-Pick disease type C (NPC) is a rare, progressive, neurodegenerative disease associated with neurovisceral manifestations resulting from lysosomal dysfunction and aberrant lipid accumulation. A multicentre, prospective observational study (Clinical Trials.gov ID: NCT02435030) of individuals with genetically confirmed NPC1 or NPC2 receiving routine clinical care was conducted, to prospectively characterize and measure NPC disease progression and to investigate potential NPC-related biomarkers versus healthy individuals. Progression was measured using the abbreviated 5-domain NPC Clinical Severity Scale (NPCCSS), 17-domain NPCCSS and NPC clinical database (NPC-cdb) score. Cholesterol esterification and heat shock protein 70 (HSP70) levels were assessed from peripheral blood mononuclear cells (PBMCs), cholestane-3ß,5α-,6ß-triol (cholestane-triol) from serum, and unesterified cholesterol from both PBMCs and skin biopsy samples. The inter- and intra-rater reliability of the 5-domain NPCCSS was assessed by 13 expert clinicians' rating of four participants via video recordings, repeated after ≥ 3 weeks. Intraclass correlation coefficients (ICCs) were calculated. RESULTS: Of the 36 individuals with NPC (2-18 years) enrolled, 31 (86.1%) completed the 6-14-month observation period; 30/36 (83.3%) were receiving miglustat as part of routine clinical care. A mean (± SD) increase in 5-domain NPCCSS scores of 1.4 (± 2.9) was observed, corresponding to an annualized progression rate of 1.5. On the 17-domain NPCCSS, a mean (± SD) progression of 2.7 (± 4.0) was reported. Compared with healthy individuals, the NPC population had significantly lower levels of cholesterol esterification (p < 0.0001), HSP70 (p < 0.0001) and skin unesterified cholesterol (p = 0.0006). Cholestane-triol levels were significantly higher in individuals with NPC versus healthy individuals (p = 0.008) and correlated with the 5-domain NPCCSS (Spearman's correlation coefficient = 0.265, p = 0.0411). The 5-domain NPCCSS showed high ICC agreement in inter-rater reliability (ICC = 0.995) and intra-rater reliability (ICC = 0.937). CONCLUSIONS: Progression rates observed were consistent with other reports on disease progression in NPC. The 5-domain NPCCSS reliability study supports its use as an abbreviated alternative to the 17-domain NPCCSS that focuses on the most relevant domains of the disease. The data support the use of cholestane-triol as a disease monitoring biomarker and the novel methods of measuring unesterified cholesterol could be applicable to support NPC diagnosis. Levels of HSP70 in individuals with NPC were significantly decreased compared with healthy individuals. TRIAL REGISTRATION: CT-ORZY-NPC-001: ClincalTrials.gov NCT02435030, Registered 6 May 2015, https://clinicaltrials.gov/ct2/show/NCT02435030 ; EudraCT 2014-005,194-37, Registered 28 April 2015, https://www.clinicaltrialsregister.eu/ctr-search/trial/2014-005194-37/DE . OR-REL-NPC-01: Unregistered.

Cerebrospinal fluid oxidative stress metabolites in patients with bipolar disorder and healthy controls: a longitudinal case-control study.

Bipolar disorder (BD) is a mental disorder characterized by recurrent relapses of affective episodes, cognitive impairment, illness progression, and reduced life expectancy. Increased systemic oxidatively generated nucleoside damage have been found in some neurodegenerative disorders and in BD. As the first, this naturalistic prospective, longitudinal follow-up case-control study investigated cerebrospinal fluid (CSF) oxidative stress markers 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) that relate to RNA and DNA damage, respectively. Patients with BD (n = 86, 51% female) and gender-and-age-matched healthy control individuals (HC; n = 44, 44% female) were evaluated at baseline (T0), during (T1) and after a new affective episode (T2), if it occurred, and after a year (T3). Cerebrospinal and urine oxidative stress markers were analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. CSF-8-oxoGuo was statistically significantly higher by 18% (p = 0.003) in BD versus HC at T0, and by 22% (p = 0) at T3. CSF-8-oxoGuo had increased by 15% (p = 0.042) from T0 to T3, and by 14% (p = 0.021) from T2 to T3 in patients, who experienced an episode during follow-up. CSF-8-oxodG had increased by 26% (p = 0.054) from T0 to T2 and decreased by 19% (p = 0.041) from T2 to T3 in patients, who experienced an episode during follow-up. CSF-8-oxoGuo did not show a statistically significant change in HC during the one-year follow-up. CSF and urine-8-oxoGuo levels correlated moderately. In conclusion, CSF oxidative stress marker of RNA damage 8-oxoGuo showed both state and trait dependence in BD and stability in HC. Central RNA damage may be a potential biomarker for BD.

HSP70 induces liver X receptor pathway activation and cholesterol reduction in vitro and in vivo.

OBJECTIVE: Heat Shock Proteins (HSPs) maintain cellular homeostasis under stress. HSP70 represents a major stress-inducible family member and has been identified as a druggable target in inherited cholesterol-sphingolipid storage diseases. We investigated if HSP70 modulates cholesterol accumulation in more common conditions related to atherogenesis. METHODS: We studied the effects of recombinant HSP70 in cholesterol-laden primary macrophages from human blood donors and pharmacological HSP70 upregulation in high-cholesterol diet fed zebrafish. RESULTS: Recombinant HSP70 facilitated cholesterol removal from primary human macrophage foam cells. RNA sequencing revealed that HSP70 induced a robust transcriptional re-programming, including upregulation of key targets of liver X receptors (LXR), master regulators of whole-body cholesterol removal. Mechanistically, HSP70 interacted with the macrophage LXRalpha promoter, increased LXRalpha and its target mRNAs, and led to elevated levels of key proteins facilitating cholesterol efflux, including ATP-binding cassette transporters A1 and G1. Pharmacological augmentation of endogenous HSP70 in high-cholesterol diet fed zebrafish activated LXR and its target mRNAs and reduced cholesterol storage at the whole organism level. CONCLUSION: These data demonstrate that HSP70 exerts a cholesterol lowering effect in primary human cells and animals and uncover a nuclear action of HSP70 in mediating cross-talk between HSP and LXR transcriptional regulation.

Animal models for Niemann-Pick type C: implications for drug discovery & development.

INTRODUCTION: Niemann-Pick type C (NPC) is a neurovisceral, progressively detrimental lysosomal storage disease with very limited therapeutic options and no approved treatment available in the US. Despite its rarity, NPC has seen increased drug developmental efforts over the past decade, culminating in the completion of two potential registration trials in 2018. Areas covered: This review highlights the many available animal models that have been developed in the field and briefly covers classical and new cell technologies. This review provides a high-level evaluation and prioritization of the various models with regard to efficient and clinically translatable drug development, and briefly discusses the relevant developments and opportunities pertaining to this. Expert opinion: With a number of in vitro and in vivo models available, and with having several drugs, all with various mechanisms of action, either approved or in late stage development, the NPC field is in an exciting time. One of the challenges for researchers and developers will be the ability to make use of the lessons learnt from existing late-stage programs as well as the incorporation not only of the opportunities but also the limitations of the many models into successful drug discovery and translational development programs.

The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase.

BACKGROUND: Gaucher Disease is caused by mutations of the GBA gene which encodes the lysosomal enzyme acid beta-glucosidase (GCase). GBA mutations commonly affect GCase function by perturbing its protein homeostasis rather than its catalytic activity. Heat shock proteins are well known cytoprotective molecules with functions in protein homeostasis and lysosomal function and their manipulation has been suggested as a potential therapeutic strategy for GD. The investigational drug arimoclomol, which is in phase II/III clinical trials, is a well-characterized HSP amplifier and has been extensively clinically tested. Importantly, arimoclomol efficiently crosses the blood-brain-barrier presenting an opportunity to target the neurological manifestations of GD, which remains without a disease-modifying therapy. METHODS: We used a range of biological and biochemical in vitro assays to assess the effect of arimoclomol on GCase activity in ex vivo systems of primary fibroblasts and neuronal-like cells from GD patients. FINDINGS: We found that arimoclomol induced relevant HSPs such as ER-resident HSP70 (BiP) and enhanced the folding, maturation, activity, and correct cellular localization of mutated GCase across several genotypes including the common L444P and N370S mutations in primary cells from GD patients. These effects where recapitulated in a human neuronal model of GD obtained by differentiation of multipotent adult stem cells. INTERPRETATION: These data demonstrate the potential of HSP-targeting therapies in GCase-deficiencies and strongly support the clinical development of arimoclomol as a potential therapeutic option for the neuronopathic forms of GD. FUNDING: The research was funded by Orphazyme A/S, Copenhagen, Denmark.

Annual severity increment score as a tool for stratifying patients with Niemann-Pick disease type C and for recruitment to clinical trials.

BACKGROUND: Niemann-Pick disease type C (NPC) is a lysosomal storage disease with a heterogeneous neurodegenerative clinical course. Multiple therapies are in clinical trials and inclusion criteria are currently mainly based on age and neurological signs, not taking into consideration differential individual rates of disease progression. RESULTS: In this study, we have evaluated a simple metric, denoted annual severity increment score (ASIS), that measures rate of disease progression and could easily be used in clinical practice. We show that ASIS is stable over several years and can be used to stratify patients for clinical trials. It achieves greater homogeneity of the study cohort relative to age-based inclusion and provides an evidence-based approach for establishing inclusion/exclusion criteria. In addition, we show that ASIS has prognostic value and demonstrate that treatment with an experimental therapy - acetyl-DL-leucine - is associated with a reduction in ASIS scores. CONCLUSION: ASIS has the potential to be a useful metric for clinical monitoring, trial recruitment, for prognosis and measuring response to therapy.

Differences in clinical presentation between bipolar I and II disorders in the early stages of bipolar disorder: A naturalistic study.

AIM: In a naturalistic clinical study of patients in the early stages of bipolar disorders the aim was to assess differences between patients with bipolar I (BD I) and bipolar II (BD II) disorders on clinical characteristics including affective symptoms, subjective cognitive complaints, functional level, the presence of comorbid personality disorders and coping strategies. METHODS: Diagnoses were confirmed using the Structured Clinical Interview for DSM-IV Disorders. Clinical symptoms were rated with the Young Mania Rating Scale and the Hamilton Depression Rating Scale, and functional status using the Functional Assessment Short Test. Cognitive complaints were assessed using the Massachusetts General Hospital Cognitive and Physical Functioning Questionnaire, the presence of comorbid personality disorders using the Standardized Assessment of Personality - Abbreviated Scale and coping style using the Coping Inventory for Stressful Situations. RESULTS: In total, 344 patients were included (BD I (n=163) and BD II (n=181). Patients with BD II presented with significantly more depressive symptoms, more cognitive complaints, lower overall functioning, and a higher prevalence of comorbid personality disorders. Finally, they exhibited a trend towards using less adaptive coping styles. LIMITATION: It cannot be omitted that some patients may have progressed from BD II to BD I. Most measures were based on patient self report. CONCLUSIONS: Overall, BD II was associated with a higher disease burden. Clinically, it is important to differentiate BD II from BD I and research wise, there is a need for tailoring and testing specific interventions towards BD II.

Heat shock protein-based therapy as a potential candidate for treating the sphingolipidoses.

Lysosomal storage diseases (LSDs) often manifest with severe systemic and central nervous system (CNS) symptoms. The existing treatment options are limited and have no or only modest efficacy against neurological manifestations of disease. We demonstrate that recombinant human heat shock protein 70 (HSP70) improves the binding of several sphingolipid-degrading enzymes to their essential cofactor bis(monoacyl)glycerophosphate in vitro. HSP70 treatment reversed lysosomal pathology in primary fibroblasts from 14 patients with eight different LSDs. HSP70 penetrated effectively into murine tissues including the CNS and inhibited glycosphingolipid accumulation in murine models of Fabry disease (Gla(-/-)), Sandhoff disease (Hexb(-/-)), and Niemann-Pick disease type C (Npc1(-/-)) and attenuated a wide spectrum of disease-associated neurological symptoms in Hexb(-/-) and Npc1(-/-) mice. Oral administration of arimoclomol, a small-molecule coinducer of HSPs that is currently in clinical trials for Niemann-Pick disease type C (NPC), recapitulated the effects of recombinant human HSP70, suggesting that heat shock protein-based therapies merit clinical evaluation for treating LSDs.

Accuracy of abdominal auscultation for bowel obstruction.

AIM: To investigate the accuracy and inter-observer variation of bowel sound assessment in patients with clinically suspected bowel obstruction. METHODS: Bowel sounds were recorded in patients with suspected bowel obstruction using a Littmann(®) Electronic Stethoscope. The recordings were processed to yield 25-s sound sequences in random order on PCs. Observers, recruited from doctors within the department, classified the sound sequences as either normal or pathological. The reference tests for bowel obstruction were intraoperative and endoscopic findings and clinical follow up. Sensitivity and specificity were calculated for each observer and compared between junior and senior doctors. Interobserver variation was measured using the Kappa statistic. RESULTS: Bowel sound sequences from 98 patients were assessed by 53 (33 junior and 20 senior) doctors. Laparotomy was performed in 47 patients, 35 of whom had bowel obstruction. Two patients underwent colorectal stenting due to large bowel obstruction. The median sensitivity and specificity was 0.42 (range: 0.19-0.64) and 0.78 (range: 0.35-0.98), respectively. There was no significant difference in accuracy between junior and senior doctors. The median frequency with which doctors classified bowel sounds as abnormal did not differ significantly between patients with and without bowel obstruction (26% vs 23%, P = 0.08). The 53 doctors made up 1378 unique pairs and the median Kappa value was 0.29 (range: -0.15-0.66). CONCLUSION: Accuracy and inter-observer agreement was generally low. Clinical decisions in patients with possible bowel obstruction should not be based on auscultatory assessment of bowel sounds.

Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities.

Lysosomes play a vital role in the maintenance of cellular homeostasis through the recycling of cell constituents, a key metabolic function which is highly dependent on the correct function of the lysosomal hydrolases and membrane proteins, as well as correct membrane lipid stoichiometry and composition. The critical role of lysosomal functionality is evident from the severity of the diseases in which the primary lesion is a genetically defined loss-of-function of lysosomal hydrolases or membrane proteins. This group of diseases, known as lysosomal storage diseases (LSDs), number more than 50 and are associated with severe neurodegeneration, systemic disease, and early death, with only a handful of the diseases having a therapeutic option. Another key homeostatic system is the metabolic stress response or heat shock response (HSR), which is induced in response to a number of physiological and pathological stresses, such as protein misfolding and aggregation, endoplasmic reticulum stress, oxidative stress, nutrient deprivation, elevated temperature, viral infections, and various acute traumas. Importantly, the HSR and its cardinal members of the heat shock protein 70 family has been shown to protect against a number of degenerative diseases, including severe diseases of the nervous system. The cytoprotective actions of the HSR also include processes involving the lysosomal system, such as cell death, autophagy, and protection against lysosomal membrane permeabilization, and have shown promise in a number of LSDs. This review seeks to describe the emerging understanding of the interplay between these two essential metabolic systems, the lysosomes and the HSR, with a particular focus on their potential as a therapeutic target for LSDs.

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein.

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-∆SBD and Hsp70-∆NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD. Association of Hsp70 with phosphatidylcholine (PC) liposomes is weak, with insertion of its Trps into the bilayer hydrocarbon region. In the presence of cardiolipin (CL), bis-monoacylglycero phosphate (BMP), or phosphatidylserine (PS) Hsp70 attaches to membranes peripherally, without penetration. Our data suggest that the organelle distribution of Hsp70 is determined by their specific lipid compositions, with Hsp70 associating with the above lipids in mitochondria, lysosomes, and the surface of cancer cells, respectively. NBD and SBD attach to lipids by extended phospholipid anchorage, with specific acidic phospholipids associating with Hsp70 in the extended conformation with acyl chains inserting into hydrophobic crevices within Hsp70, and other chains remaining in the bilayer. This anchorage is expected to cause a stringent orientation of Hsp70 on the surface. Our data further suggest that acidic phospholipids induce a transition of SBD into the molten globule state, which may be essential to allow SBD-substrate interaction also within the hydrophobic bilayer interior acyl chain region.

Effects of a screening and treatment protocol with haloperidol on post-cardiotomy delirium: a prospective cohort study.

OBJECTIVES: Post-cardiotomy delirium is common and associated with increased morbidity and mortality. No gold standard exists for detecting delirium, and evidence to support the choice of treatment is needed. Haloperidol is widely used for treating delirium, but indication, doses and therapeutic targets vary. Moreover, doubt has been raised regarding overall efficacy. The purpose of this study was to assess the effect of a combination of early detection and standardized treatment with haloperidol on post-cardiotomy delirium, with the hypothesis that the proportion of delirium- and coma-free days could be increased. Length of stay (LOS), complications and 180-day mortality are reported. METHODS: Prospective interventional cohort study. One hundred and seventeen adult patients undergoing cardiac surgery were included before introduction of a screening and treatment protocol with haloperidol for delirium, and 123 patients were included after. Nurses screened patients using validated tools (the Delirium Observation Screening (DOS) scale and confusion assessment method for the intensive care unit (CAM-ICU)). In case of delirium, a checklist to eliminate precipitating/ inducing factors and a protocol for standardized dosing with haloperidol was applied. Group comparison was done using non-parametric tests and analysis of fractions, and associations between delirium and predefined covariates were analysed with logistic regression. RESULTS: Incidence of delirium after cardiac surgery was 21 (14-29) and 22 (15-30) %, onset was on postoperative day 1 (1-4) and 1 (1-3), duration was 1 (1-4) day and 3 (1-5) days, respectively, with no significant difference (Period 1 vs 2, all values are given as the median and 95% confidence interval). The proportion of delirium- and coma-free days was 67 (61-73) and 65 (60-70) %, respectively (ns). There was no difference in LOS or complication rate. Delirium was associated to increasing age, increased length of stay and complications. CONCLUSIONS: We observed no increase in the proportion of delirium- and coma-free days after introduction of a combination of early detection and standardized treatment with haloperidol on post-cardiotomy delirium. Most patients were not severely affected, and the few who were, proved difficult to treat, indicating that a simple treatment protocol with haloperidol was ineffective.

Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase.

Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert multidrug resistance. Their cancer selectivity is associated with transformation-associated reduction in ASM expression and subsequent failure to maintain sphingomyelin hydrolysis during drug exposure. Taken together, these data identify ASM as an attractive target for cancer therapy.

Sunitinib and SU11652 inhibit acid sphingomyelinase, destabilize lysosomes, and inhibit multidrug resistance.

Defective apoptosis signaling and multidrug resistance are major barriers for successful cancer treatment. To identify drugs capable of targeting treatment-resistant cancer cells, we screened small-molecule kinase inhibitor libraries for compounds that decrease the viability of apoptosis-resistant human MCF7-Bcl-2 breast cancer cells. SU11652, a multitargeting receptor tyrosine kinase inhibitor, emerged as the most potent compound in the screen. In addition to MCF7-Bcl-2 cells, it effectively killed HeLa cervix carcinoma, U-2-OS osteosarcoma, Du145 prostate carcinoma, and WEHI-S fibrosarcoma cells at low micromolar concentration. SU11652 accumulated rapidly in lysosomes and disturbed their pH regulation and ultrastructure, eventually leading to the leakage of lysosomal proteases into the cytosol. Lysosomal destabilization was preceded by an early inhibition of acid sphingomyelinase, a lysosomal lipase that promotes lysosomal membrane stability. Accordingly, Hsp70, which supports cancer cell survival by increasing lysosomal acid sphingomyelinase activity, conferred partial protection against SU11652-induced cytotoxicity. Remarkably, SU11652 killed multidrug-resistant Du145 prostate cancer cells as effectively as the drug-sensitive parental cells, and subtoxic concentrations of SU11652 effectively inhibited multidrug-resistant phenotype in Du145 prostate cancer cells. Notably, sunitinib, a structurally almost identical and widely used antiangiogenic cancer drug, exhibited similar lysosome-dependent cytotoxic activity, albeit with significantly lower efficacy. The significantly stronger lysosome-targeting activity of SU11652 suggests that it may display better efficacy in cancer treatment than sunitinib, encouraging further evaluation of its anticancer activity in vivo. Furthermore, our data provide a rationale for novel approaches to target drug-resistant cancers by combining classic chemotherapy with sunitinib or SU11652.

Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens.

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.