The promise of epigenome-wide association studies and cancer-specific somatic DNA methylation changes in improving our understanding of cancer, coupled with the decreasing cost and increasing coverage of DNA methylation microarrays, has brought about a surge in the use of these technologies. Here, we aim to provide both a review of issues encountered in the processing and analysis of array-based DNA methylation data and a summary of the advantages of recent approaches proposed for handling those issues, focusing on approaches publicly available in open-source environments such as R and Bioconductor. We hope that the processing tools and analysis flowchart described herein will facilitate researchers to effectively use these powerful DNA methylation array-based platforms, thereby advancing our understanding of human health and disease.
DNA methylation; microarray; processing; analysis; bioconductor and R packages
DNA methylation profiles can be used to define molecular cancer subtypes that may better inform disease etiology and clinical decision-making. This investigation aimed to create DNA methylation profiles of bladder cancer based on CpG methylation from almost 800 cancer-related genes and to then examine the relationship of those profiles with exposures related to risk and clinical characteristics. DNA, derived from formalin-fixed paraffin-embedded tumor samples obtained from incident cases involved in a population-based case-control study of bladder cancer in New Hampshire, was used for methylation profiling on the Illumina GoldenGate Methylation Bead Array. Unsupervised clustering of those loci with the greatest change in methylation between tumor and non-diseased tissue was performed to defined molecular subgroups of disease, and univariate tests of association followed by multinomial logistic regression was used to examine the association between these classes, bladder cancer risk factors and clinical phenotypes. Membership in the two most methylated classes was significantly associated with invasive disease (P < 0.001 for both class 3 and 4). Male gender (P = 0.04) and age >70 years (P = 0.05) was associated with membership in one of the most methylated classes. Finally, average water arsenic levels in the highest percentile predicted membership in an intermediately methylated class of tumors (P = 0.02 for both classes). Exposures and demographic associated with increased risk of bladder cancer specifically associate with particular subgroups of tumors defined by DNA methylation profiling and these subgroups may define more aggressive disease.
Simian virus-40 (SV40) is a DNA tumour virus that was introduced into the human population with contaminated poliovirus vaccine, and its role in mesothelioma is widely debated. PCR based testing has been called into question, as false positives can be because of cross-reactivity with related viruses, or to laboratory contamination. The Institute of Medicine has recommended the development of more sensitive and specific tests to resolve this controversy.
We have characterized highly sensitive RT–PCR based assays that are specific for SV40-encoded microRNAs (miRNAs), as an alternative to current testing methods.
Using this sensitive and specific detection method, we were unable to identify SV40 miRNA expression in human malignant pleural mesothelioma (MM) samples.
Our work indicates that SV40 miRNAs are not likely to contribute to mesothelioma tumourogenesis, but highlights the value of this approach when compared with the relatively unspecific current testing methods.
mesothelioma; microRNA; SV40
Several studies have revealed increased bone mineral density (BMD) in patients with knee or hip osteoarthritis, but few studies have addressed this issue in hand osteoarthritis (HOA). The aims of this study were to compare BMD levels and frequency of osteoporosis between female patients with HOA, rheumatoid arthritis (RA) and controls aged 50–70 years, and to explore possible relationships between BMD and disease characteristics in patients with HOA.
190 HOA and 194 RA patients were recruited from the respective disease registers in Oslo, and 122 controls were selected from the population register of Oslo. All participants underwent BMD measurements of femoral neck, total hip and lumbar spine (dual‐energy x ray absorptiometry), interview, clinical joint examination and completed self‐reported questionnaires.
Age‐, weight‐ and height‐adjusted BMD values were significantly higher in HOA versus RA and controls, the latter only significant for femoral neck and lumbar spine. The frequency of osteoporosis was not significantly different between HOA and controls, but significantly lower in HOA versus RA. Adjusted BMD values did not differ between HOA patients with and without knee OA, and significant associations between BMD levels and symptom duration or disease measures were not observed.
HOA patients have a higher BMD than population‐based controls, and this seems not to be limited to patients with involvement of larger joints. The lack of correlation between BMD and disease duration or severity does not support the hypothesis that higher BMD is a consequence of the disease itself.
Little is known about signaling pathways, besides those of neurotrophic factors, that are operational in adult spiral ganglion neurons. In patients with sensorineural hearing loss, such pathways could eventually be targeted to stimulate and guide neurite outgrowth from the remnants of the spiral ganglion towards a cochlear implant, thereby improving the fidelity of sound transmission. To systematically identify neuronal receptors for guidance cues in the adult cochlea, we conducted a genome-wide cDNA microarray screen with two-month-old CBA/CaJ mice. A meta-analysis of our data and those from older mice in two other studies revealed the presence of neuronal transmembrane receptors that represent all four established guidance pathways—ephrin, netrin, semaphorin, and slit—in the mature cochlea as late as 15 months. In addition, we observed the expression of all known receptors for the Wnt morphogens, whose neuronal guidance function has only recently been recognized. In situ hybridizations located the mRNAs of the Wnt receptors frizzled 1, 4, 6, 9, and 10 specifically in adult spiral ganglion neurons. Finally, frizzled 9 protein was found in the growth cones of adult spiral ganglion neurons that were regenerating neurites in culture. We conclude from our results that adult spiral ganglion neurons are poised to respond to neurite damage, owing to the constitutive expression of a large and diverse collection of guidance receptors. Wnt signaling, in particular, emerges as a candidate pathway for guiding neurite outgrowth towards a cochlear implant after sensorineural hearing loss.
Cell surface receptors; Cochlea; In situ hybridization; Mice; Microarray analysis
The novel synergistic drug candidate CRx-102 comprises dipyridamole and low dose prednisolone and is in clinical development for the treatment of immunoinflammatory diseases. The purpose of this clinical study was to examine the efficacy and safety of CRx-102 in patients with hand osteoarthritis (HOA).
The study was conducted as a blinded, randomised, placebo-controlled trial at four centres in Norway. Eligibility criteria included being of age 30–70 years, at least one swollen and tender joint, a Kellgren–Lawrence (K–L) score of 2 or higher on radiographs, and a score of at least 30 mm pain on the Australian/Canadian Osteoarthritis Hand Index (AUSCAN) visual analogue pain scale (VAS). The primary endpoint was a reduction in pain from baseline to day 42 on the AUSCAN pain subscale. Two-sided p values for the differences in least squares (LS) means adjusted for baseline are presented.
The mean age of the 83 patients with HOA was 60 years and 93% were females. CRx-102 was statistically superior to placebo at 42 days for changes in AUSCAN pain (LS mean −14.2 vs −4.0) and for clinically relevant secondary endpoints (joint pain VAS (−18.6 vs −6.3), patient global VAS (−15.9 vs −4.2)) in the intention to treat population. The most frequently reported adverse event during the study was headache (52% in CRx-102 vs 15% in the placebo group).
The novel synergistic drug candidate CRx-102 demonstrated efficacy by statistically reducing pain compared to placebo in HOA and was generally well tolerated.
Sindbis virus expression vectors have been used successfully to express and silence genes of interest in vivo in several mosquito species, including Aedes aegypti, Ae. albopictus, Ae. triseriatus,Culex pipiens, Armigeres subalbatus and Anopheles gambiae. Here we describe the expression of an endogenous gene, defensin, in Ae. aegypti using the orally infectious Sindbis virus, MRE/3′2J expression vector. We optimized conditions to infect mosquito larvae per os using C6/36 Ae. albopictus cells infected with the recombinant virus to maximize virus infection and expression of defensin. Infection with the parental Sindbis virus (MRE/3′2J) did not induce defensin expression. Mosquito larvae infected by ingestion of recombinant Sindbis virus-infected C6/36 cells expressed defensin when they emerged as adults. Defensin expression was observed by western analysis or indirect fluorescent assay in all developmental stages of mosquitoes infected with MRE/3′2J virus that contained the defensin insert. The multiplicity of infection of C6/36 cells and the quantity of infected cells consumed by larvae played an important role in defensin expression. Parental viruses, missing the defensin insert, and/or other defective interfering virus may have contributed to these observations.
Sindbis virus; defensin; gene expression; MRE/3'2J; mosquito
We have recently introduced 125I-hCG as an elimination marker in patients with human chorionic gonadotrophin (hCG) producing testicular cancer. 125I-hCG is a well-known reagent in clinical biochemistry and is used extensively in hCG assays. Previous studies have shown that the iodination process leaves the hCG molecule mainly intact. The iodination, purification and stability of 125I-hCG tracer are described. The aim of the present study was to determine whether or not 125I is associated with hCG after the injection of 125I-hCG intravenously (i.v.) in humans. Three different methods were used. Following injection of 125I-hCG, the plasma disappearance of radioactivity and hCG were followed for a period of 28 days in 13 normal subjects. Serum from a normal healthy male following injection of 125I-hCG was analysed using a double antibody direct binding radioimmunoassay specific for holo-hCG and high performance liquid chromatography (HPLC). Following injection of 125I-hCG in eight normal healthy males and five normal healthy females, the disappearance of radioactivity and hCG showed identical paths in the 28 days follow-up period. The bindable radioactive fraction of immunologically active hCG in serum of a normal healthy male following injection of 125I-hCG was between 57.0% and 72.1%, and was constant over time. HPLC showed similar elution pattern of serum from a normal healthy male injected i.v. with 125I-hCG and 125I-hCG. Using three different methods, we were able concurrently to demonstrate the association of 125I with hCG in humans up to 28 days after injection of radiolabelled hCG i.v. Thus, information about the expected elimination of hCG can be obtained by following the elimination of activity in plasma after injection of 125I-hCG. © 1999 Cancer Research Campaign
gonadotropins; human chorionic; radioiodinated hCG; in vivo elimination
The rate of reduction in the concentration of serum human chorionic gonadotrophin (hCG) following chemotherapy for germ cell tumours may follow a complex pattern, with longer apparent half-life during later stages of chemotherapy, even in patients treated successfully. The commonly used half-life of less than 3 days for hCG to monitor the effect of chemotherapy in patients with germ cell tumours of the testis may represent too simple a model. 125I-labelled hCG was injected intravenously in 27 patients with germ cell tumours and elevated hCG during chemotherapy. The plasma radioactivity and hCG concentrations were followed. During chemotherapy, the plasma disappearance of hCG showed a biphasic pattern, with an initial fast and a later slow component in all patients. Using the steep part of the hCG plasma disappearance curve, five patients who achieved long-term remission had half-lives longer than 3 days (3.6–6.8 days), whereas four out of five patients not achieving long-term remission had half-lives shorter than 3 days. After the third treatment cycle, eight patients who achieved long-term remission had hCG half-lives longer than 3 days (7.4–17.0 days). In these patients, the plasma disappearance of [125I]hCG was equivalent to that of hCG. Thus, the slow decline of hCG represented a slow plasma disappearance rather than a hCG production from vital tumour cells and could, consequently, not be used to select patients for additional or intensified chemotherapy. The concept of a fixed half-life for plasma hCG during treatment of hCG-producing germ cell tumours is inappropriate and should be revised. Difficulties in interpreting a slow decline of hCG may be overcome by comparing the plasma disappearance of total hCG with the plasma disappearance of [125I]hCG. © 1999 Cancer Research Campaign
gonadotrophin; human chorionic; testicular neoplasm; chemotherapy
The predominant genetic defect causing p47-phox-deficient chronic granulomatous disease (A47 degrees CGD) is a GT deletion (DeltaGT) at the beginning of exon 2. No explanation exists to account for the high incidence of this single mutation causing a rare disease in an unrelated, racially diverse population. In each of 34 consecutive unrelated normal individuals, both the normal and mutant DeltaGT sequences were present in genomic DNA, suggesting that a p47-phox related sequence carrying DeltaGT exists in the normal population. Screening of genomic bacteriophage and YAC libraries identified 13 p47-phox bacteriophage and 19 YAC clones. The GT deletion was found in 11 bacteriophage and 15 YAC clones. Only 5 exonic and 33 intronic differences distinguished all DeltaGT clones from all wild-type clones. The most striking differences were a 30-bp deletion in intron 1 and a 20-bp duplication in intron 2. These results provide good evidence for the existence of at least one highly homologous p47-phox pseudogene containing the DeltaGT mutation. The p47-phox gene and pseudogene(s) colocalize to chromosome 7q11.23. This close linkage, together with the presence within each gene of multiple recombination hot spots, suggests that the predominance of the DeltaGT mutation in A47 degrees CGD is caused by recombination events between the wild-type gene and the pseudogene(s).
Our previous studies have shown that licochalcone A, an oxygenated chalcone, has antileishmanial (M. Chen, S.B. Christensen, J. Blom, E. Lemmich, L. Nadelmann, K. Fich, T.G. Theander, and A. Kharazmi, Antimicrob, Agents Chemother. 37:2550-2556, 1993; M. Chen, S.B. Christensen, T.G. Theander, and A. Khrazmi, Antimicrob. Agents Chemother. 38:1339-1344, 1994) and antimalarial (M. Chen, T.G. Theander, S.B. Christensen, L. Hviid, L. Zhai, and A. Kaharazmi, Antimicrob. Agents Chemother. 38:1470-1475, 1994) activities. We have observed that licochalcone A alters the ultrastructure of the mitochondria of Leishmania promastigotes (Chen et al., Antimicrob. Agents Chemother. 37:2550-2556, 1993). The present study was designed to examine this observation further and investigate the mechanism of action of antileishmanial activity of licochalcone A. Electron microscopic studies showed that licochalcone A altered the ultrastructure of Leishmania major promastigote and amastigote mitochondria in a concentration-dependent manner without damaging the organelles of macrophages or the phagocytic function of these cells. Studies on the function of the parasite mitochondria showed that licochalcone A inhibited the respiration of the parasite by the parasites. Moreover, licochalcone A inhibited the activity of the parasite mitochondrial dehydrogenase. The inhibition of the activity of the parasite mitochondrial enzyme correlated well with the changes in the ultrastructure of the mitochondria shown by electron microscopy. These findings demonstrate that licochalcone A alters the ultrastructure and function of the mitochondria of Leishmania parasites.
Licochalcone A, isolated from Chinese licorice roots, inhibited the in vitro growth of both chloroquine-susceptible (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains in a [3H]hypoxanthine uptake assay. The growth inhibition of the chloroquine-resistant strain by licochalcone A was similar to that of the chloroquine-susceptible strain. To examine the activity of licochalcone A on the different asexual blood stages of the parasite, licochalcone A was added to highly synchronized cultures containing rings, trophozoites, and schizonts. The growth of the parasites at all stages was inhibited by licochalcone A. The in vivo activity of licochalcone A was tested in a mouse model of infection with P. yoelii. Licochalcone A administered either intraperitoneally or orally for 3 to 6 days protected the mice from the otherwise lethal P. yoelii infection. These results demonstrate that licochalcone A exhibits potent antimalarial activity and might be developed into a new antimalarial drug.
This study was designed to examine the antileishmanial activity of the oxygenated chalcone licochalcone A in mice and hamsters infected with Leishmania parasites. Intraperitoneal administration of licochalcone A at doses of 2.5 and 5 mg/kg of body weight per day completely prevented lesion development in BALB/c mice infected with Leishmania major. Treatment of hamsters infected with L. donovani with intraperitoneal administration of licochalcone A at a dose of 20 mg/kg of body weight per day for 6 consecutive days resulted in a > 96% reduction of parasite load in the liver and the spleen compared with values for untreated control animals. The [3H]thymidine uptake by the parasites isolated from the treated hamsters was only about 1% of that observed in parasites isolated from the controls. Oral administration of licochalcone A at concentrations of 5 to 150 mg/kg of body weight per day for 6 consecutive days resulted in > 65 and 85% reductions of L. donovani parasite loads in the liver and the spleen, respectively, compared with those of untreated control hamsters. These data clearly demonstrate that licochalcone A is a promising lead for the development of a new drug against leishmaniases.
Licochalcone A, an oxygenated chalcone isolated from the roots of Chinese licorice plant, inhibited the growth of both Leishmania major and Leishmania donovani promastigotes and amastigotes. The structure of the licochalcone A was established by mass and nuclear magnetic resonance spectroscopies and by synthesis, and its purity was verified by high-pressure liquid chromatography. The 50% inhibition of growth of logarithmic- and stationary-phase promastigotes of L. major, as measured by [3H]thymidine uptake, were 4 and 2.5 micrograms/ml, respectively. The growth of L. major promastigotes was totally inhibited after a 20-h incubation period with licochalcone A at 5 micrograms/ml. At a concentration of 0.5 microgram/ml, licochalcone A markedly reduced the infection rate of human peripheral blood monocyte-derived macrophages and U937 cells with L. major promastigotes and exhibited a strong intracellular killing of the parasite. These data show that intracellular Leishmania amastigotes are more susceptible than promastigotes to licochalcone A. Results of studies on the site of action of licochalcone A indicate that the target organelle appears to be the parasite mitochondria. These findings demonstrate that licochalcone A in concentrations that are nontoxic to host cells exhibits a strong antileishmanial activity and that appropriate substituted chalcones might be a new class of antileishmanial drugs.
Restriction fragment length polymorphism (RFLP) analysis, comparative marker chromosome analysis, and polymorphic enzyme analysis was carried out on a total of eight human urothelial cell lines and sublines selected according to our knowledge of their HLA-A,B phenotype. RFLP analysis and cytogenetic analysis showed that the cell lines Hu1703He, Hu1922, and T24 are genuine cell lines of different origin. The identity of Hu1703He could not be confirmed by its isozyme phenotype which was identical to the T24 phenotype. RFLP analysis and isozyme analysis revealed that three cell lines, Hu456, Hu549, and Hu961a, and two transformed sublines, HCV-29Tmv and Hu609Tmv, are sublines of T24. A common origin of Hu456, Hu549, Hu961a, HCV-29Tmv, and Hu609Tmv was confirmed by marker chromosome analysis. However, the T24 origin of these cytogenetically related cell lines was not supported by chromosome analysis of T24. RFLP analysis and HLA phenotyping of two tumorigenic and invasive sublines isolated from a culture of non-tumorigenic Hu609 cells showed that non-tumorigenic Hu609 cells can transform 'spontaneously' in vitro into tumorigenic Hu609T cells. The results emphasise the need for careful monitoring and screening of cell lines for their identity using more than one identification parameter.
OBJECTIVE--To devise and evaluate a rapid screening method for detecting trisomy 21 (Down's syndrome) in samples of uncultured amniotic fluid cells. DESIGN--Non-radioactive in situ hybridisation with HY128, a 500,000 base pair yeast artificial chromosome probe specific for chromosome 21. Blinded study of 12 karyotypically normal amniotic fluid samples and eight samples trisomic for chromosome 21. SETTING--Cytogenetic and obstetric services at a tertiary referral centre, Copenhagen. MAIN OUTCOME MEASURES--Time necessary to complete the test. Proportion of cell nuclei containing two and three hybridisation signals in karyotypically normal and abnormal amniotic fluid samples. RESULTS--The test could be completed within three to four days after amniocentesis. In the normal samples a mean of 73% (range 61-82%) of the amniotic cell nuclei showed two hybridisation signals and 6% (0-18%) showed three signals. By contrast, among the trisomic samples 29% (19-38%) of the nuclei exhibited two signals and 48% (31-60%) showed three signals. CONCLUSION--The technique clearly distinguished between normal and trisomic samples. Prenatal diagnosis with in situ hybridisation with chromosome specific probes was fast and may make it possible to screen for selected, aneuploidies. However, the technique is still at a preliminary stage and needs further evaluation and refinement.
1. At concentrations above 10 ng ml-1, the tumour promoter thapsigargin stimulates the release of radioactivity from [3H]-arachidonic acid-labelled macrophages harvested from rat peritoneal cavity. 2. The release of radioactivity from prelabelled macrophages was augmented more than additively when the cells were incubated in the medium containing both thapsigargin (10 ng ml-1) and other tumour promoters (10 ng ml-1), such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin. 3. Thapsigargin required extracellular Ca2+ for the stimulation of arachidonic acid release, while TPA did not. 4. Cytoplasmic free calcium level was increased by thapsigargin treatment but not by TPA treatment. 5. An inhibitor of protein kinases, H-7 inhibited the effect of TPA dose-dependently, whereas H-7 did not inhibit that of thapsigargin. 6. These results suggest that thapsigargin stimulates arachidonic acid release by a mechanism different from that of TPA, viz by acting as a selective Ca2+ mobilizer, but not by activating protein kinase C as TPA does.
The ability of thapsigargin and thapsigargicin to activate mast cells and leukocytes has been investigated. The thapsigargin-induced histamine release from rat peritoneal mast cells was found to be dependent on the concentration of thapsigargin, the purity of the mast cell preparations, and the number of mast cells in suspension. Thapsigargin induced histamine release from human basophil leukocytes. Thapsigargin induced beta-glucuronidase and lysozyme release from human neutrophil leukocytes. Thapsigargin caused a release of histamine from mesentery, lung, and heart mast cells of the rat, but only to a minor extent from the corresponding guinea-pig cells. Thapsigargicin induced histamine release from mesentery, lung, and heart mast cells of the rat at concentrations from 0.1 microM but provoked only a release from the corresponding guinea-pig cells in the concentration-range 0.16 to 1.6 microM. Thapsigargin increased the cytoplasmic free calcium level in intact human blood platelets at concentrations from 3.0 nM.