Genome-wide association study of obsessive-compulsive disorder.

Obsessive-compulsive disorder (OCD) is a common, debilitating neuropsychiatric illness with complex genetic etiology. The International OCD Foundation Genetics Collaborative (IOCDF-GC) is a multi-national collaboration established to discover the genetic variation predisposing to OCD. A set of individuals affected with DSM-IV OCD, a subset of their parents, and unselected controls, were genotyped with several different Illumina SNP microarrays. After extensive data cleaning, 1465 cases, 5557 ancestry-matched controls and 400 complete trios remained, with a common set of 469,410 autosomal and 9657 X-chromosome single nucleotide polymorphisms (SNPs). Ancestry-stratified case-control association analyses were conducted for three genetically-defined subpopulations and combined in two meta-analyses, with and without the trio-based analysis. In the case-control analysis, the lowest two P-values were located within DLGAP1 (P=2.49 × 10(-6) and P=3.44 × 10(-6)), a member of the neuronal postsynaptic density complex. In the trio analysis, rs6131295, near BTBD3, exceeded the genome-wide significance threshold with a P-value=3.84 × 10(-8). However, when trios were meta-analyzed with the case-control samples, the P-value for this variant was 3.62 × 10(-5), losing genome-wide significance. Although no SNPs were identified to be associated with OCD at a genome-wide significant level in the combined trio-case-control sample, a significant enrichment of methylation QTLs (P<0.001) and frontal lobe expression quantitative trait loci (eQTLs) (P=0.001) was observed within the top-ranked SNPs (P<0.01) from the trio-case-control analysis, suggesting these top signals may have a broad role in gene expression in the brain, and possibly in the etiology of OCD.

Genome-wide association study of Tourette's syndrome.

Tourette's syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (P<5 × 10(-8)); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (P=1.85 × 10(-6)). A secondary analysis including an additional 211 cases and 285 controls from two closely related Latin American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (P=3.6 × 10(-7) for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder.

Frequent occurrence of multidrug-resistant CC17 Enterococcus faecium among clinical isolates in Sweden.

AIMS: To screen for the globally spread cluster of Enterococcus faecium, clonal complex 17 (CC17) and characterize the genetic profile of Swedish clinical Ent. faecium isolates. METHODS: A total of 203 consecutive isolates collected from 2004 to 2007 from patients with bacteraemia in Sweden. All isolates were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA) and 20 isolates representing different MLVA types (MT) were chosen for multilocus sequence typing (MLST). Minimal inhibitory concentrations against clinically relevant antibiotics were determined with agar dilution. Presence of the virulence genes esp and hyl was investigated using PCR. RESULTS: A total of 65% (n = 109) of all isolates belonged to MT-1, and the second most common MLVA type was MT-159 (13%, n = 21). MLST analysis confirmed the presence of CC17 during the entire study period. The number of isolates resistant to gentamicin and vancomycin, as well as the presence of hyl, increased significantly during the investigation period. CONCLUSIONS: The present study demonstrates that nosocomial infections caused by Ent. faecium CC17 are commonly occurring in Sweden. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of CC17 Ent. faecium in Sweden. The increase of antibiotic resistance and virulence indicates that these strains are further adapting to the hospital environment.

Validating genetic risk associations for ovarian cancer through the international Ovarian Cancer Association Consortium.

The search for genetic variants associated with ovarian cancer risk has focused on pathways including sex steroid hormones, DNA repair, and cell cycle control. The Ovarian Cancer Association Consortium (OCAC) identified 10 single-nucleotide polymorphisms (SNPs) in genes in these pathways, which had been genotyped by Consortium members and a pooled analysis of these data was conducted. Three of the 10 SNPs showed evidence of an association with ovarian cancer at P< or =0.10 in a log-additive model: rs2740574 in CYP3A4 (P=0.011), rs1805386 in LIG4 (P=0.007), and rs3218536 in XRCC2 (P=0.095). Additional genotyping in other OCAC studies was undertaken and only the variant in CYP3A4, rs2740574, continued to show an association in the replication data among homozygous carriers: OR(homozygous(hom))=2.50 (95% CI 0.54-11.57, P=0.24) with 1406 cases and 2827 controls. Overall, in the combined data the odds ratio was 2.81 among carriers of two copies of the minor allele (95% CI 1.20-6.56, P=0.017, p(het) across studies=0.42) with 1969 cases and 3491 controls. There was no association among heterozygous carriers. CYP3A4 encodes a key enzyme in oestrogen metabolism and our finding between rs2740574 and risk of ovarian cancer suggests that this pathway may be involved in ovarian carcinogenesis. Additional follow-up is warranted.

Increased conjugation frequencies in clinical Enterococcus faecium strains harbouring the enterococcal surface protein gene esp.

This study compared the in-vitro ability of Enterococcus faecium isolates of different origin to acquire vanA by conjugation in relation to the occurrence of the esp gene. In total, 29 clinical isolates (15/29 esp+), 30 normal intestinal microflora isolates (2/30 esp+) and one probiotic strain (esp-) were studied with a filter-mating assay. Conjugation events were confirmed by PCR and pulsed-field gel electrophoresis. Among the infection-derived isolates, the esp+ isolates had higher conjugation frequencies compared with esp- isolates (p < 0.001), with a median value of 6.4 x 10(-6) transconjugants/donor. The probiotic strain was shown to acquire vanA vancomycin resistance in in-vitro filter mating experiments.

Effects of mevinolin treatment on tissue dolichol and ubiquinone levels in the rat.

Rats were treated with mevinolin by intraperitoneal injection (15 days) or dietary administration (30 days). The cholesterol, dolichol, dolichyl phosphate and ubiquinone contents of the liver, brain, heart, muscle and blood were then investigated. The cholesterol contents of these organs did not change significantly, with the exception of muscle. Intraperitoneal administration of the drug increases the amount of dolichol in liver, muscle and blood and decreases the dolichyl-P amount in muscle. The same treatment increases the level of ubiquinone in muscle and blood and decreases this value in liver and heart. Oral administration decreases dolichol, dolichyl-P and ubiquinone levels in heart and muscle, while in liver the dolichol level is elevated and ubiquinone level lowered. In brain the amount of dolichyl-P is increased. Intraperitoneal injection of mevinolin also modifies the liver dolichol and dolichyl-P isoprenoid pattern, with an increase in shorter chain polyisoprenes. The levels of dolichol and ubiquinone in the blood do not follow the changes observed in other tissues. Incorporation of [3H]acetate into cholesterol by liver slices prepared from mevinolin-treated rats exhibited an increase, whereas in brain no change was seen. Labeling of dolichol and ubiquinone was increased in both liver and brain, but incorporation into dolichyl phosphate remained relatively stable. The results indicate that mevinolin affects not only HMG-CoA reductase but, to some extent, also affects certain of the peripheral enzymes, resulting in considerable effects on the various mevalonate pathway lipids.

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices.

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.

Age-dependent modifications in the metabolism of mevalonate pathway lipids in rat brain.

The levels and rates of biosynthesis of mevalonate pathway lipids in rat brain were investigated during development and aging. Between birth and 18 months of age there are only moderate decreases in the phospholipid and cholesterol contents but an increase in the levels of dolichyl-P and, particularly of dolichol. The amount of ubiquinone is unchanged. The rate of incorporation of [3H]leucine into protein decreases by 10% during the first year, while the incorporation of [3H]glycerol into phospholipids decreases by 20%. The high rates of [3H]mevalonate incorporation into cholesterol and dolichol after birth decreases rapidly. In contrast, the rate of incorporation into ubiquinone is constant. Squalene synthase activity decreases rapidly in the early postnatal period and at 18 months of age this activity is 10-fold lower than immediately after birth. cis-Prenyltransferase activity is also high during the first postnatal month and reaches a constant level at 4 months of age. Significantly, nonaprenyl 4-hydroxybenzoate transferase activity is high during the entire period investigated. The rate of lipid peroxidation does not change during aging. These results demonstrate that brain cholesterol and dolichol exhibit a low rate of turnover during aging, whereas ubiquinone is synthesized at a high rate and exhibits rapid turnover throughout the entire lifespan.

Effect of antimicrobial agents on the ecological balance of human microflora.

The normal microflora acts as a barrier against colonisation of potentially pathogenic microorganisms and against overgrowth of already present opportunistic microorganisms. Control of growth of opportunistic microorganisms is termed colonisation resistance. Administration of antimicrobial agents, therapeutically or as prophylaxis, causes disturbances in the ecological balance between the host and the normal microflora. Most studies on the impact of antimicrobial agents on normal microflora have been carried out on the intestinal flora. Less is known on the effects on oropharyngeal, skin, and vaginal microflora. Disturbances in the microflora depend on the properties of the agents as well as of the absorption, route of elimination, and possible enzymatic inactivation and/or binding to faecal material of the agents. The clinically most common disturbances in the intestinal microflora are diarrhoea and fungal infections that usually cease after the end of treatment. A well-balanced microflora prevents establishment of resistant microbial strains. By using antimicrobial agents that do not disturb colonisation resistance, the risk of emergence and spread of resistant strains between patients and dissemination of resistant determinants between microorganisms is reduced. In this article, the potential ecological effects of administration of antimicrobial agents on the intestinal, oropharyngeal, and vaginal microflora are summarised. The review is based on clinical studies published during the past 10 years.

Effect on the human normal microflora of oral antibiotics for treatment of urinary tract infections.

Oral administration of antibiotics for treatment of urinary tract infections (UTIs) can cause ecological disturbances in the normal intestinal microflora. Poorly absorbed drugs can reach the colon in active form, suppress susceptible microorganisms and disturb the ecological balance. Suppression of the normal microflora may lead to reduced colonization resistance with subsequent overgrowth of pre-existing, naturally resistant microorganisms, such as yeasts and Clostridium difficile. New colonization by resistant potential pathogens may also occur and may spread within the body or to other patients and cause severe infections. It is therefore important to learn more about the ecological effects of antibacterial agents on the human microflora. The impact on intestinal microorganisms of oral antibiotics used for the treatment of UTIs is reviewed here. Ampicillin, amoxycillin and co-amoxiclav suppress both the aerobic and anaerobic intestinal microflora with overgrowth of ampicillin-resistant Enterobacteriaceae. Pivmecillinam also affects the intestinal microflora, suppressing Escherichia coli, but does not have a major effect on the anaerobic microflora. Several orally administered cephalosporins, such as cefixime, cefpodoxime, cefprozil and ceftibuten, reduce the number of Enterobacteriaceae and increase the number of enterococci. Colonization with C. difficile has also been observed. Fluoroquinolones eliminate or strongly suppress intestinal Enterobacteriaceae, but affect enterococci and anaerobic bacteria only slightly. When antimicrobial agents are prescribed for the treatment of UTIs, not only the antimicrobial spectrum of the agent but also the potential ecological disturbances, including the risk of emergence of resistant strains, should be considered.

Effect of quinolones on intestinal ecology.

Quinolones have a selective effect on the normal human intestinal microflora. Published data on 13 different quinolone agents [ciprofloxacin, enoxacin, norfloxacin, ofloxacin, pefloxacin, lomefloxacin, levofloxacin, sparfloxacin, rufloxacin, sitafloxacin (DU-6859a), gatifloxacin, trovafloxacin and moxifloxacin] show that gram-negative aerobic bacteria, especially Enterobacteriaceae, are strongly suppressed or eliminated during therapy. Gram-positive aerobic cocci are affected strongly by administration of sitafloxacin and moxifloxacin and to minor degrees by the other quinolones. Three new quinolones--gatifloxacin, trovafloxacin and moxifloxacin--are very active against anaerobic bacteria in vitro but have minor effects on the anaerobic intestinal human microflora. Similar findings have been reported for the other 10 quinolones. Thus, the quinolone antibacterials have an ecological impact on the human intestinal microflora, mainly on the enterobacteria, that should be taken into account when these agents are used for prophylaxis or treatment of gastrointestinal bacterial infections.

Effects of prolonged administration of phthalate ester on the liver.

Di(2-ethylhexyl)phthalate (DEHP) was administered to male rats in the diet at concentrations of 2.0, 0.2, and 0.02% for up to 102 weeks. Low doses resulted in moderate increases in certain hepatic enzymes during the initial phase of exposure and in a continuous increase in the activities of these same enzymes throughout the treatment period. An increased level of dolichol and decreased concentration of dolichyl-P were observed. Furthermore, the rate of protein glycosylation diminished. Liver biopsies from patients subjected to hemodialysis demonstrated an increased number of peroxisomes. Phthalate ester seems to interfere with protein turnover, so that the half-life of total mitochondrial and microsomal protein is considerably increased.

Isoprenoids in aging and neurodegeneration.

During aging the human brain shows a progressive increase in levels of dolichol, a reduction in levels of ubiquinone, but relatively unchanged concentrations of cholesterol and dolichyl phosphate. In a neurodegenerative disease, Alzheimer's disease, the situation is reversed with decreased levels of dolichol and increased levels of ubiquinone. The concentrations of dolichyl phosphate are also increased, while cholesterol remains unchanged. This study shows that the isoprenoid changes in Alzheimer's disease differ from those occurring during normal aging and that this disease cannot, therefore, be regarded as a result of premature aging. The increase in the sugar carrier dolichyl phosphate may reflect an increased rate of glycosylation in the diseased brain and the increase in the endogenous anti-oxidant ubiquinone an attempt to protect the brain from oxidative stress, for instance induced by lipid peroxidation.

Content and fatty acid composition of cardiolipin in the brain of patients with Alzheimer's disease.

The frontal, temporal and occipital cortex from human brains affected by Alzheimer's disease were analyzed for their contents and fatty acid compositions of cardiolipin. Phospholipids were purified using an HPLC system and cardiolipin was found to be present in the same amount (on a protein basis) as in age-matched controls. One-third of the total fatty acyl moieties of this phospholipid were saturated, one-third monounsaturated and one-third polyunsaturated. In affected brain regions the levels of certain polyunsaturated fatty acids displayed moderate decreases, not exceeding 10-15%. However, the total amount of polyunsaturated fatty acids decreased by only 9%. These results demonstrate that the amount and structure of brain cardiolipin are not modified to any great extent in connection with Alzheimer's disease.

Trypanosomes cause dysregulation of c-fos expression in the rat suprachiasmatic nucleus.

Rats infected with the parasite Trypanosoma brucei brucei showed selective changes of c-fos expression in the suprachiasmatic nucleus of the hypothalamus (SCN) during spontaneous sleep (S) and wakefulness (W) under a basal 12 h/12 h light-dark (L-D) cycle. In the vast majority of W (D phase) control animals the SCN was devoid of cells displaying Fos-related immunopositivity, while Fos-like-immunoreactive (ir) neurones were detected in most S (L phase) control rats. In most infected animals, on the other hand, Fos-ir neurones were detected in the SCN during W, but not during the S period, with a significant difference between control and infected S rats. Thus, these data indicate that the basal c-fos expression in the SCN during the L-D and S-W cycles is considerably altered in experimental trypanosomiasis. This is the first observation of a selective change in the SCN in trypanosome-infected rat brains. Since the SCN plays an important role as a pace-maker for biological rhythms, this finding may provide a basis for understanding the pathogenesis behind endogenous rhythm dyregulation and changes in sleeping pattern in human trypanosomiasis (African sleeping sickness).

Interferon-gamma and a factor derived from trypanosomes cause behavioural changes in the rat.

A newly isolated interferon-gamma (IFN-gamma) immunoreactive molecule, "neuronal IFN-gamma", and recombinant lymphocyte-derived IFN-gamma were injected intracerebroventricularly (i.c.v.) through a previously implanted cannula into adult male rats during both the light and the dark phases of the light/dark cycle. The two molecules caused a reduction in both frequency and duration of rearing and locomotion during the dark, but not the light, phase. A molecule isolated from Trypanosoma brucei brucei, a parasite of the same subspecies of trypanosomes which causes African sleeping sickness, can induce production and release of IFN-gamma and "neuronal IFN-gamma" from lymphocytes and neurons, respectively. I.c.v. injection of this factor also reduced rearing during the dark period, but to a less extent. Thus, "neuronal IFN-gamma" appears to have effects on animal behaviour in common with lymphocyte-derived IFN-gamma. This study highlights the potential role of these cytokines in behaviour disturbances.

Mechanism of quinolone resistance in anaerobic bacteria.

Several recently developed quinolones have excellent activity against a broad range of aerobic and anaerobic bacteria and are thus potential drugs for the treatment of serious anaerobic and mixed infections. Resistance to quinolones is increasing worldwide, but is still relatively infrequent among anaerobes. Two main mechanisms, alteration of target enzymes (gyrase and topoisomerase IV) caused by chromosomal mutations in encoding genes, or reduced intracellular accumulation due to increased efflux of the drug, are associated with quinolone resistance. These mechanisms have also been found in anaerobic species. High-level resistance to the newer broad-spectrum quinolones often requires stepwise mutations in target genes. The increasing emergence of resistance among anaerobes may be a consequence of previous widespread use of quinolones, which may have enriched first-step mutants in the intestinal tract. Quinolone resistance in the Bacteroides fragilis group strains is strongly correlated with amino acid substitutions at positions 82 and 86 in GyrA (equivalent to positions 83 and 87 of Escherichia coli). Several studies have indicated that B. fragilis group strains possess efflux pump systems that actively expel quinolones, leading to resistance. DNA gyrase seems also to be the primary target for quinolones in Clostridium difficile, since amino acid substitutions in GyrA and GyrB have been detected in resistant strains. To what extent other mechanisms, such as mutational events in other target genes or alterations in outer-membrane proteins, contribute to resistance among anaerobes needs to be further investigated.

Anaerobic bacteria commonly colonize the lower airways of intubated ICU patients.

OBJECTIVES: To investigate respiratory tract colonization by aerobic and anaerobic bacteria in mechanically ventilated patients. METHODS: Bacterial colonization of the stomach and the respiratory tract was qualitatively and quantitatively analyzed over time in 41 consecutive mechanically ventilated patients in a Swedish intensive care unit (ICU), with special emphasis on elucidation of the role of anaerobic bacteria in the lower respiratory tract. Samples were taken from the oropharynx, gastric juice, subglottic space and trachea within 24 h (median 14 h) of intubation, and then every third day until day 18 and every fifth day until day 33. RESULTS: The patients were often heavily colonized with microorganisms not considered to belong to a healthy normal oropharyngeal and gastric flora on admission to the ICU. A majority harbored enterococci, coagulase-negative staphylococci and Candida spp. in at least one site on day 1. Anaerobic bacteria, mainly peptostreptococci and Prevotella spp., were isolated from subglottic and/or tracheal secretions in 59% of the patients. Different routes of tracheal colonization for different groups of microorganisms were found. Primary or concomitant colonization of the oropharynx with staphylococci, enterococci, enterobacteria and Candida was often seen, while Pseudomonas spp., other non-fermenting Gram-negative rods and several anaerobic species often primarily colonized the trachea, indicating exogenous or direct gastrointestinal routes of colonization. CONCLUSIONS: Mechanically ventilated patients were heavily colonized in their lower airways by potential pathogenic microorganisms, including a high load of anaerobic bacteria. Different routes of colonization were shown for different species.

Ecological effects on the oro- and nasopharyngeal microflora in children after treatment of acute otitis media with cefuroxime axetil or amoxycillin-clavulanate as suspensions.

OBJECTIVE: To evaluate if the extent of normal microflora disturbances differed between treatment with amoxycillin-clavulanate administered in an active form and cefuroxime axetil administered as an inactive prodrug. METHODS: Twenty-eight children, 0.5-5 years old, diagnosed with acute otitis media (AOM), were treated with either amoxycillin-clavulanate (13.3 mg/kg 3 times daily) or cefuroxime axetil (15 mg/kg twice daily) for 7 days. Saliva samples and nasopharyngeal swabs were collected before, directly after and 2 weeks after treatment. The saliva samples were quantitatively and qualitatively analyzed and the nasopharyngeal swabs were qualitatively analyzed. All isolated strains were tested for beta-lactamase production. RESULTS: Both treatment regimens gave rise to similar alterations of the normal oropharyngeal microflora. In both groups, the amount of Streptococcus salivarius was significantly reduced (P < 0.05). The most common causative pathogens of acute otitis were S. pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. On the day of enrollment, approximately half of the patients, in both groups, were infected with more than one pathogen. The rate of infection or colonization with more than one potential pathogen was low on day 7 but recurred 2 weeks after treatment to similar levels as on day 0. The total number of patients with reinfection, recolonization or recurrence of pathogens on day 21 was 11/12 in the amoxycillin-clavulanate group and 4/7 in the cefuroxime axetil group. The most common beta-lactamase producer was M. catarrhalis. CONCLUSION: The local high concentration of antibiotics in the oropharynx immediately after intake of antibiotic suspensions seem to have little or no impact on the extent of disturbance of the microflora in this region. Children of this age group seem prone to either reinfection, recolonization or persistence of pathogens within 2 weeks after treatment. Furthermore, co-infection with more than one pathogen seems common in children with AOM and infection with beta-lactamase producing microorganisms occurs frequently.

Changes in hepatic dolichol and dolichyl monophosphate caused by treatment of rats with inducers of the endoplasmic reticulum and peroxisomes and during ontogeny.

Rats were treated with various inducers of the endoplasmic reticulum and peroxisomes and the properties and distributions of dolichol and dolichyl phosphate analyzed. The treatment of rats with carcinogenic agents 2-acetylaminofluorene, N-nitrosodiethylamine and 3-methylcholanthrene and with the compounds such as phenobarbital, terpentine, cholestyramine and di(2-ethylhexyl)phthalate have all caused changes in the microsomal or lysosomal contents of dolichol to various extents, but only the latter group influenced dolichyl-P concentration. Shortly after birth, the hepatic content of dolichyl-P reaches the adult level, whereas the level of the free alcohol is low at birth but increases continuously thereafter. Incorporation of [3H]mevalonate into dolichol was also dependent on factors other than de novo synthesis, e.g., the pool size. Rates of glycosylation reactions dependent on dolichyl-P exhibit considerable changes but are independent of the existing levels of lipid intermediate. GDP-mannosyl transferase activity increases greatly with birth, but the enzyme activity returns to the adult level within a day after birth. These results demonstrate that structural and functional modifications induced with drugs can greatly influence the content and distribution of dolichol which are independent of the existing levels of dolichyl-P.