Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

Performance Characteristics of the BluePrint® Breast Cancer Diagnostic Test.

The analytical performance of a multi-gene diagnostic signature depends on many parameters, including precision, repeatability, reproducibility and intra-tumor heterogeneity. Here we study the analytical performance of the BluePrint 80-gene breast cancer molecular subtyping test through determination of these performance characteristics. BluePrint measures the expression of 80 genes that assess functional pathways which determine the intrinsic breast cancer molecular subtypes (i.e. Luminal-type, HER2-type, Basal-type). Knowing a tumor's dominant functional pathway can help allocate effective treatment to appropriate patients. Here we show that BluePrint is a highly precise and highly reproducible test with correlations above 98% based on the generated index and subtype concordance above 99%. Therefore, BluePrint can be used as a robust and reliable tool to identify breast cancer molecular subtypes.

Decentralization of Next-Generation RNA Sequencing-Based MammaPrint® and BluePrint® Kit at University Hospitals Leuven and Curie Institute Paris.

A previously developed and centrally validated MammaPrint® (MP) and BluePrint® (BP) targeted RNA next-generation sequencing (NGS) kit was implemented and validated in two large academic European hospitals. Additionally, breast cancer molecular subtypes by MP and BP RNA sequencing were compared with immunohistochemistry (IHC). Patients with early breast cancer diagnosed at University Hospitals Leuven and Curie Institute Paris were prospectively included between September 2017 and January 2018. Formalin-fixed paraffin-embedded tissue sections were analyzed with MP and BP NGS technology at the beta sites and with both NGS and microarray technology at Agendia. Raw NGS data generated on Illumina MiSeq instruments at the beta sites were interpreted and compared with NGS and microarray data at Agendia. MP and BP NGS molecular subtypes were compared to surrogate IHC breast cancer subtypes. Equivalence of MP and BP indices was determined by Pearson's correlation coefficient. Acceptable limits were defined a priori, based on microarray data generated at Agendia between 2012 and 2016. The concordance, the Negative Percent Agreement and the Positive Percent Agreement were calculated based on the contingency tables and had to be equal to or higher than 90%. Out of 124 included samples, 48% were MP Low and 52% High Risk with microarray. Molecular subtypes were BP luminal, HER2 or basal in 82%, 8% and 10% respectively. Concordance between MP microarray at Agendia and MP NGS at the beta sites was 91.1%. Concordance of MP High and Low Risk classification between NGS at the beta sites and NGS at Agendia was 93.9%. Concordance of MP and BP molecular subtyping using NGS at the beta sites and microarray at Agendia was 89.5%. Concordance between MP and BP NGS subtyping, and IHC was 71.8% and 76.6%, for two IHC surrogate models. The MP/BP NGS kit was successfully validated in a decentralized setting.

MammaPrint and BluePrint Molecular Diagnostics Using Targeted RNA Next-Generation Sequencing Technology.

Next-generation DNA sequencing is rapidly becoming an indispensable tool for genome-directed cancer diagnostics, but next-generation RNA sequencing (RNA-seq) is currently not standardly used in clinical diagnostics for expression assessment. However, multigene RNA diagnostic assays are used increasingly in the routine diagnosis of early-stage breast cancer. Two of the most widely used tests are currently available only as a central laboratory service, which limits their clinical use. We evaluated the use of RNA-seq as a decentralized method to perform such tests. The MammaPrint and BluePrint RNA-seq tests were found to be equivalent to the clinically validated microarray tests. The RNA-seq tests were highly reproducible when performed in different locations and were stable over time. The MammaPrint RNA-seq test was clinically validated. Our data demonstrate that RNA-seq can be used as a decentralized platform, yielding results substantially equivalent to results derived from the predicate diagnostic device.

Generation of 3 human induced pluripotent stem cell lines LUMCi005-A, B and C from a Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type patient.

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch type (HCHWA-D) is an autosomal dominant hereditary disease caused by a point mutation in exon 17 of the APP gene. We generated human induced pluripotent stem cells (hiPSCs) from a symptomatic HCHWA-D patient by using non-integrating Sendai virus (SeV). The newly generated hiPSCs express all pluripotency markers, have a normal karyotype, carry the Dutch mutation, can differentiate in the three germ layers in vitro and are SeV free.

An observational study on disturbed peripheral circadian rhythms in hemodialysis patients.

The quality of life of hemodialysis (HD) patients is hampered by reduced nocturnal sleep quality and excessive daytime sleepiness. In addition to the sleep/wake cycle, levels of circadian biomarkers (e.g. melatonin) are disturbed in end-stage renal disease (ESRD). This suggests impaired circadian clock performance in HD patients, but the underlying mechanism is unknown. In this observational study, diurnal rhythms of sleep, serum melatonin and cortisol concentrations and clock gene mRNA expression are compared between HD patients (n = 9) and healthy control subjects (n = 9). In addition, the presence of circulating factors that might affect circadian rhythmicity is tested in vitro with cell culture experiments. Reduced sleep quality (median sleep onset latency [interquartile range] of 23.9 [17.3] min for patients versus 5.0 [10] minutes for controls, p < 0.01; mean (± SD) sleep efficiency 70.2 ± 8.1% versus 82.9 ± 10.9%, p = 0.02 and mean awake minutes after sleep onset 104.8 ± 27.9 versus 54.6 ± 41.6 minutes, p = 0.01) and increased daytime sleepiness (mean Epworth Sleepiness Score of 10.0 ± 4.8 versus 3.9 ± 2.0, p < 0.01) were confirmed in HD patients. Reduced nocturnal melatonin concentrations (1 AM: 98.1 [122.9] pmol/L versus 12.5 [44.2] pmol/L, p = 0.019; 5 AM: 114.0 [131.6] pmol/L versus 11.8 [86.8] pmol/L, p = 0.031) and affected circadian control of cortisol rhythm and circadian expression of the clock gene REV-ERBα were found. HD patient serum had a higher capacity to synchronize cells in vitro, suggesting an accumulated level of clock resetting compounds in HD patients. These compounds were not cleared by hemodialysis treatment or related to frequently used medications. In conclusion, the abovementioned results strongly suggest a disturbance in circadian timekeeping in peripheral tissues of HD patients. Accumulation of clock resetting compounds possibly contributes to this. Future studies are needed for a better mechanistic understanding of the interaction between renal failure and perturbation of the circadian clock.

Long-term relationship between methylphenidate and tobacco consumption and nicotine craving in adults with ADHD in a prospective cohort study.

Patients with Attention-Deficit/Hyperactivity disorder (ADHD) have higher smoking rates, a younger age of smoking onset, and increased difficulty to stop smoking as compared to controls. Methylphenidate induced acute effects of increased smoking in laboratory studies, but long-term effects are unknown. We studied the acute and long-term relationship between methylphenidate use and tobacco consumption and nicotine craving among ADHD patients naïve for methylphenidate (N=325). Patients filled out the Smoking Questionnaire (SQ) at baseline, and after two-weeks and three-months of methylphenidate use. The SQ involved questions on demographics, tobacco consumption, nicotine craving, life events, psychiatric diagnoses and use of medication. At baseline, smoking prevalence of ADHD patients was twice as high (50.2%) as the national norm (25.6%; p<.001). Tobacco consumption increased with 1.3 cigarettes per day after three-months of methylphenidate use. When translated into pack years, tobacco consumption increased by about 23 packs per year. Reports of increased nicotine craving after methylphenidate, increased with 20.3% after two weeks and 29.2% after three months. Light smokers (1-12 cigarettes/day) were especially at risk for increased tobacco consumption (p<.05). Thus although methylphenidate is the drug of choice in medical treatment for ADHD, tobacco consumption and nicotine craving increased acutely and stabilized at increased levels after three-months of methylphenidate use. Although the net effect of methylphenidate on smoking behavior and craving should be further investigated within a randomized, placebo-controlled design, the results suggest that active prevention of increased smoking is needed in patients prescribed methylphenidate.

A gene expression profile for detection of sufficient tumour cells in breast tumour tissue: microarray diagnosis eligibility.

BACKGROUND: Microarray diagnostics of tumour samples is based on measurement of prognostic and/or predictive gene expression profiles. Typically, diagnostic profiles have been developed using bulk tumour samples with a sufficient amount of tumour cells (usually >50%). Consequentially, a diagnostic results depends on the minimal percentage of tumour cells within a sample. Currently, tumour cell percentage is assessed by conventional histopathological review. However, even for experienced pathologists, such scoring remains subjective and time consuming and can lead to ambiguous results. METHODS: In this study we investigated whether we could use transcriptional activity of a specific set of genes instead of histopathological review to identify samples with sufficient tumour cell content. Genome-wide gene expression measurements were used to develop a transcriptional gene profile that could accurately assess a sample's tumour cell percentage. RESULTS: Supervised analysis across 165 breast tumour samples resulted in the identification of a set of 13 genes which expression correlated with presence of tumour cells. The developed gene profile showed a high performance (AUC 0.92) for identification of samples that are suitable for microarray diagnostics. Validation on 238 additional breast tumour samples indicated a robust performance for correct classification with an overall accuracy of 91 percent and a kappa score of 0.63 (95%CI 0.47-0.73). CONCLUSION: The developed 13-gene profile provides an objective tool for assessment whether a breast cancer sample contains sufficient tumour cells for microarray diagnostics. It will improve the efficiency and throughput for diagnostic gene expression profiling as it no longer requires histopathological analysis for initial tumour percentage scoring. Such profile will also be very use useful for assessment of tumour cell percentage in biopsies where conventional histopathology is difficult, such as fine needle aspirates.

An immune response enriched 72-gene prognostic profile for early-stage non-small-cell lung cancer.

PURPOSE: Current staging methods are imprecise for predicting prognosis of early-stage non-small-cell lung cancer (NSCLC). We aimed to develop a gene expression profile for stage I and stage II NSCLC, allowing identification of patients with a high risk of disease recurrence within 2 to 3 years after initial diagnosis. EXPERIMENTAL DESIGN: We used whole-genome gene expression microarrays to analyze frozen tumor samples from 172 NSCLC patients (pT1-2, N0-1, M0) from five European institutions, who had undergone complete surgical resection. Median follow-up was 89 months (range, 1.2-389) and 64 patients developed a recurrence. A random two thirds of the samples were assigned as the training cohort with the remaining samples set aside for independent validation. Cox proportional hazards models were used to evaluate the association between expression levels of individual genes and patient recurrence-free survival. A nearest mean analysis was used to develop a gene-expression classifier for disease recurrence. RESULTS: We have developed a 72-gene expression prognostic NSCLC classifier. Based on the classifier score, patients were classified as either high or low risk of disease recurrence. Patients classified as low risk showed a significantly better recurrence-free survival both in the training set (P < 0.001; n = 103) and in the independent validation set (P < 0.01; n = 69). Genes in our prognostic signature were strongly enriched for genes associated with immune response. CONCLUSIONS: Our 72-gene signature is closely associated with recurrence-free and overall survival in early-stage NSCLC patients and may become a tool for patient selection for adjuvant therapy.

Influence of the fungal NF-kappaB inhibitor panepoxydone on inflammatory gene expression in MonoMac6 cells.

The fungal secondary metabolite panepoxydone has been recently described as an inhibitor of NF-kappaB activation which is a pivotal regulator of the inflammatory and immune response. These findings have led to propose that panepoxydone may be useful as anti-inflammatory agent. In this study we investigated for the first time the effects of panepoxydone on inflammatory gene expression in the monocytic cell line MonoMac6, stimulated with lipopolysaccharide (LPS) and the phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA). DNA microarray analysis of 110 human genes known to be strongly regulated during inflammation, combined with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) revealed that low micromolar concentrations (12-24 microM) of panepoxydone strongly inhibited the expression of thirty-three NF-kappaB dependent pro-inflammatory genes such as the chemokines CCL3, CCL4, CCL8; CXCL8, CXCL10, CXCL20, the cytokines IL-1, IL-6, TNF-alpha, pro-inflammatory enzymes like COX-2, and components of the REL/NF-kappaB/IkappaB family without significant effects on the expression of house-keeping genes. Panepoxydone strongly inhibited hTNF-alpha, IL-8 and NF-kappaB promoter activity in LPS/TPA stimulated MonoMac6 cells with IC(50) values of 0.5-1 microg/ml by blocking the phosphorylation of IkappaB and subsequent binding of the activated NF-kappaB transcription factor to the DNA. From our data, panepoxydone may serve as lead structure for the development of transcription-based inhibitors of pro-inflammatory gene expression.

Converting a breast cancer microarray signature into a high-throughput diagnostic test.

BACKGROUND: A 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis. RESULTS: To facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 probes suitable for high throughput processing. RNA of 162 patient samples from two previous studies was subjected to hybridization to this custom array to validate the prognostic value. Classification results obtained from the original analysis were then compared to those generated using the algorithms based on the custom microarray and showed an extremely high correlation of prognosis prediction between the original data and those generated using the custom mini-array (p < 0.0001). CONCLUSION: In this report we demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool. The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is therefore an excellent tool to predict outcome of disease in breast cancer patients.

Gene expression profiling in follicular lymphoma to assess clinical aggressiveness and to guide the choice of treatment.

Follicular lymphoma (FL) is a disease characterized by a long clinical course marked by frequent relapses that vary in clinical aggressiveness over time. Therefore, the main dilemma at each relapse is the choice for the most effective treatment for optimal disease control and failure-free survival while at the same time avoiding overtreatment and harmful side effects. The selection for more aggressive treatment is currently based on histologic grading and clinical criteria; however, in up to 30% of all cases these methods prove to be insufficient. Using supervised classification on a training set of paired samples from patients who experienced either an indolent or aggressive disease course, a gene expression profile of 81 genes was established that could, with an accuracy of 100%, distinguish low-grade from high-grade disease. This profile accurately classified 93% of the FL samples in an independent validation set. Most important, in a third series of FL cases where histologic grading was ambiguous, precluding meaningful morphologic guidance, the 81-gene profile shows a classification accuracy of 94%. The FL stratification profile is a more reliable marker of clinical behavior than the currently used histologic grading and clinical criteria and may provide an important alternative to guide the choice of therapy in patients with FL both at presentation and at relapse.

Laschiatrion, a new antifungal agent from a Favolaschia species (Basidiomycetes) active against human pathogens.

Laschiatrion (1), a new antifungal antibiotic, was isolated from fermentations of Favolaschia sp. 87129. (1) exhibits broad in vitro activity against several human pathogens while no antibacterial and cytotoxic activities could be detected. The structure was elucidated by spectroscopic techniques. As to our knowledge laschiatrion possesses a new steroid skeleton.

Gene expression profiles of primary breast tumors maintained in distant metastases.

It has been debated for decades how cancer cells acquire metastatic capability. It is unclear whether metastases are derived from distinct subpopulations of tumor cells within the primary site with higher metastatic potential, or whether they originate from a random fraction of tumor cells. Here we show, by gene expression profiling, that human primary breast tumors are strikingly similar to the distant metastases of the same patient. Unsupervised hierarchical clustering, multidimensional scaling, and permutation testing, as well as the comparison of significantly expressed genes within a pair, reveal their genetic similarity. Our findings suggest that metastatic capability in breast cancer is an inherent feature and is not based on clonal selection.

Gene expression profiling predicts clinical outcome of breast cancer.

Breast cancer patients with the same stage of disease can have markedly different treatment responses and overall outcome. The strongest predictors for metastases (for example, lymph node status and histological grade) fail to classify accurately breast tumours according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third; however, 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies. Here we used DNA microarray analysis on primary breast tumours of 117 young patients, and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis' signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). In addition, we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.

Mariannaepyrone--a new inhibitor of thromboxane A2 induced platelet aggregation.

Mariannaeapyrone ((E)-2-(1,3,5,7-tetramethyl-5-nonenyl)-3,5-dimethyl-6-hydroxy-4H-pyran-4-one) is a new fungal metabolite isolated from fermentations of the common mycophilic deuteromycete Mariannaea elegans. The chemical structure of the 4-pyrone was determined by spectroscopic techniques. Mariannaeapyrone is a selective inhibitor of the thromboxane A2 induced aggregation of human platelets, whereas only weak cytotoxic and antimicrobial effects could be observed.

Coprinol, a new antibiotic cuparane from a Coprinus species.

Coprinol, a new antibacterial cuparane, was isolated from fermentations of a Coprinus sp. Its biological activities were investigated and its structure was elucidated by spectroscopic methods. The new antibiotic exhibited activitiy against multidrug-resistant Gram-positive bacteria in vitro. Two derivatives were synthesized and their activities compared to the parent compound.

Inhibition of interleukin-6 signaling by galiellalactone.

A search for inhibitors of the IL-6-mediated signal transduction in HepG2 cells using secreted alkaline phosphatase (SEAP) as reporter gene resulted in the isolation of galiellalactone (1) from fermentations of the ascomycete strain A111-95. Galiellalactone inhibits the IL-6-induced SEAP expression with IC(50) values of 250-500 nM by blocking the binding of the activated Stat3 dimers to their DNA binding sites without inhibiting the tyrosine and serine phosphorylation of the Stat3 transcription factor. Due to its selective activity, galiellalactone may serve as a lead compound for the development of new therapeutic agents for diseases originating from the inappropriate expression of IL-6 and as molecular tool to dissect the JAK/STAT pathways.

Inhibition of interleukin-6 signaling and Stat3 activation by a new class of bioactive cyclopentenone derivatives.

The IL-6-dependent activation of the JAK/STAT pathway plays a central role in the induction of the acute phase response in the liver. In a search for new inhibitors of the IL-6-mediated signal transduction in HepG2 cells using secreted alkaline phosphatase (SEAP) as reporter gene, four novel cyclopentenones, 2-(1-chloropropenyl)-4,5-dihydroxycyclopent-2-enone (CPDHC, 1), 4, 5-dihydroxy-2-propenylcyclopent-2-enone (DHPC, 2), 5-hydroxy-2, 3-dimethylcyclopent-2-enone (HDC, 3), and 4-methyl-5-methylenecyclopent-3-en-1,2-diol (MMCD, 4) were isolated from fermentations of the ascomycete strain A23-98. CPDHC (1) inhibits the IL-6-induced SEAP expression with IC(50) values of 4. 0-5.3 microM (0.75-1 microg/ml). The compounds DHPC (2), HDC (3), and MMCD (4) which are structurally closely related to CPDHC (1) showed no inhibitory effects on the IL-6-induced SEAP expression in HepG2 cells. Studies on the mode of action revealed that CPDHC (1) affects the IL-6-dependent pathway by inhibiting the tyrosine phosphorylation of the STAT3 and STAT1 as well as the serine phosphorylation of the Stat3 transcription factor. In addition, CPDHC (1) and DHPC (2) inhibit the AP-1 and NF-kappaB mediated SEAP expression in transiently transfected HeLa S3 cells with IC(50) values of 10-15 microM (2-3 microg/ml) and 50-100 microM (8-16 microg/ml) respectively. Our results indicate that CPDHC inhibits the NF-kappaB pathway by preventing the phosphorylation and proteolytic degradation of the IkappaBalpha protein. The novel cyclopentenones may represent lead compounds for the development of new anti-inflammatory drugs.