Estrogen receptor-related receptor-alpha (ERR-alpha) is dysregulated in inflammatory arthritis.

OBJECTIVES: Subchondral bone loss is a characteristic feature of inflammatory arthritis. Recently, estrogen receptor-related receptor-alpha (ERR-alpha), an orphan nuclear receptor, has been found to be involved in activation of macrophages. We hypothesized that ERR-alpha which is expressed and also functional in articular chondrocytes, osteoblasts and osteoclasts, may be involved in rodent models of inflammatory arthritis. METHODS: Erosive arthritis was induced in DBA/1 mice by injection of type II collagen in Freund's complete adjuvant. RNA was isolated from the bone and joints and expression of ERR-alpha and cartilage (GDF5 and Col2a1) and bone [bone sialoprotein (BSP) and osteocalcin (OCN)] markers was analysed by semi-quantitative PCR. RESULTS: We report for the first time that the expression of ERR-alpha is dysregulated in bones and joints in a mouse model of inflammatory arthritis. Specifically, we show that ERR-alpha expression is down-regulated early in bone and later in joints of mice with type II CIA. Concomitantly, temporal changes were observed in GDF-5 and Col2a1 expression in joints following both initial injection and booster injection of type II collagen. Similarly, down-regulation of ERR-alpha mRNA expression in subchondral bone in mice with induced joint inflammation was also paralleled by down-regulation of markers of bone formation (BSP, OCN). CONCLUSIONS: These data suggest that dysregulation of ERR-alpha expression may precede and contribute to the destruction of cartilage and bone accompanying inflammatory arthritis.

Effects of a series of triorganotins on ATP levels in human natural killer cells.

Natural killer (NK) cells are our initial immune defense against viral infections and cancer development. Thus, agents that are able to interfere with their function increase the risk of cancer and/or infection. A series of triorganotins, (trimethyltin (TMT), dimethylphenyltin (DMPT), methyldiphenyltin (MDPT), and triphenyltin (TPT)) have been shown to decrease the lytic function of human NK cells. TPT and MDPT were much more effective than DMPT or TMT at reducing lytic function. This study investigates the role that decreased ATP levels may play in decreases in the lytic function of NK cells induced by these OTs. A 24 h exposure to as high as 10 muM TMT caused no decrease in ATP levels even though this level of TMT caused a greater than 75% loss of lytic function. TPT at 200 nM caused a decrease in ATP levels of about 20% while decreasing lytic function by greater than 85%. There was no association between ATP levels and lytic function for any of the compounds when NK cells were exposed for 1h or 24 h. However, after a 48 h exposure to both DMPT and TPT decreased lytic function was associated with decreased ATP levels. There was an association between decreased lytic function and decreased ATP levels after a 6 day exposure to each of the four compounds. These studies indicate that the loss of lytic function seen after 1 h and 24 h exposures to this series of organotins cannot be accounted for by decreases in ATP. However, after longer exposures loss of lytic function may be in part be attributable to inadequate ATP levels.

Association study of the nicotinic acetylcholine receptor alpha4 subunit gene, CHRNA4, in attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a common childhood-onset psychiatric condition with a strong genetic component. Evidence from pharmacological, clinical and animal studies has suggested that the nicotinic system could be involved in the disorder. Previous studies have implicated the nicotinic acetylcholine receptor alpha4 subunit gene, CHRNA4, in ADHD. Particularly, a polymorphism in the exon 2-intron 2 junction of CHRNA4 has been associated with severe inattention defined by latent class analysis. In the current study, we used the transmission disequilibrium test (TDT) to investigate four polymorphisms encompassing this region of CHRNA4 for association with ADHD in a sample of 264 nuclear families from Toronto. No significant evidence of biased transmission was observed for any of the marker alleles for ADHD defined as a categorical trait (all subtypes included), although one haplotype showed marginal evidence of under-transmission. No association was found with the ADHD predominantly inattentive subtype or with symptom dimension scores of inattention. On the contrary, nominally significant evidence of association of individual markers was obtained for the ADHD combined subtype and with teacher-rated hyperactivity-impulsivity scores, with the same haplotype being under-transmitted. Based on our results and others, CHRNA4 may be involved in ADHD; however, its role in ADHD symptomatology remains to be clarified.

Association of the dopamine receptor D1 gene, DRD1, with inattention symptoms in families selected for reading problems.

Twin studies have provided evidence for shared genetic influences between attention-deficit/hyperactivity disorder (ADHD) and specific reading disabilities (RD), with this overlap being highest for the inattentive symptom dimension of ADHD. Previously, we found evidence for association of the dopamine receptor D1 gene (DRD1) with ADHD, and with the inattentive symptom dimension in particular. This, combined with evidence for working memory (WM) deficits in individuals with RD or ADHD, and the importance of D1 receptors in attentional processes and WM function, suggests that DRD1 may be a common genetic influence underlying both disorders. Here, in a study of 232 families ascertained through probands with reading problems, we tested for association of the DRD1 gene with RD, as a categorical trait, and with quantitative measures of key reading component skills, WM ability, and inattentive symptoms. Although no associations were found with RD, or with reading component skills or verbal WM, we found evidence for association with inattentive behaviour. Specifically, DRD1 Haplotype 3, the haplotype previously found to be associated with inattentive symptoms in ADHD, is also associated with parent- and teacher-reported symptoms of inattention in this sample selected for reading problems (P=0.023 and 0.004, respectively). Together, the replicated finding of Haplotype 3 association with inattentive symptoms in two independent study samples strongly supports a role for DRD1 in attentional ability. Furthermore, the association of DRD1 with inattention, but not with RD, or the other reading and reading-related phenotypes analysed, suggests that DRD1 contributes uniquely to inattention, without overlap for reading ability.

Effect of a series of triorganotins on the immune function of human natural killer cells.

Natural killer (NK) cells are our initial immune defense against viral infections and cancer development. They are able to destroy tumor and virally infected cells. Thus, agents that are able to interfere with their function increase the risk of cancer and/or infection. Organotins (OTs) have been shown to interfere with the tumor-destroying function of human NK cells. The purpose of the current study was to explore the relationship of a series of triorganotins, that differ in structure by only a single organic group, for their capacity to block NK tumor-cell destroying (lytic) function. Here we examine the series: trimethyltin (TMT), dimethylphenyltin (DMPT), methyldiphenyltin (MDPT), and triphenyltin (TPT). NK cells were exposed to TMT, DMPT, MDPT or TPT for 1, 24, 48h, or 6d. A 1h exposure to TMT, at concentrations as high as 20µM, had no effect on lytic function. However, concentrations as low as 2.5µM were able to decrease NK tumor-destroying function after 6d. A 1h exposure to DMPT had no effect on lytic function, however, after 6d there was an 80-90% decrease in lytic function at 1µM. Exposure to MDPT (as low as 2.5µM) decreased NK function at 1h, after 6d there was as much as a 90% decrease at concentrations as low as 100nM MDPT. TPT decreased lytic function in a manner similar to MDPT, however, it was more effective at 1h than MDPT. The effect of the triorganotins on the ability of NK cells to bind to targets was studied, to determine if this contributed to the loss of lytic function. The relative immunotoxic potential of this series of compounds is TPT≈MDPT>DMPT>TMT.

Association of the calcyon gene (DRD1IP) with attention deficit/hyperactivity disorder.

Attention deficit/hyperactivity disorder (ADHD) is a childhood-onset disorder characterized by marked inattention, hyperactivity and impulsivity. The dopaminergic system has been hypothesized to be involved in the development of ADHD. Positive associations have been found for the dopamine receptors D1 and D5 genes, suggesting that other genes involved in D1/D5 signalling may also contribute to ADHD. In this study, we tested the calcyon gene (DRD1IP), which encodes a brain-specific D1-interacting protein involved in D1/D5 receptors calcium signalling, for association with ADHD. The inheritance of nine polymorphisms in the calcyon gene was examined in a sample of 215 nuclear families, with 260 affected children, using the transmission/disequilibrium test. The most common haplotype, designated C1, demonstrated significant evidence for excess transmission. Quantitative trait analyses of this haplotype showed significant relationships with both the inattentive (parent's rating, P=0.006; teacher's rating, P=0.003) and hyperactive/impulsive (parent's rating, P=0.004) dimensions of the disorder. Two of the nine marker alleles included in haplotype C1, rs4838721A located approximately 10 kb 5' of the gene and rs2275723C located 10 bp upstream of the exon 5 acceptor splice site, also showed significant evidence for association when analysed individually. As these two variants are not predicted to alter calcyon function, we screened the gene exons by sequencing. No variation in the coding region was identified, suggesting that a causal variant allele resides elsewhere in a regulatory sequence of the gene. These findings support the proposed involvement of the calcyon gene in ADHD and implicate haplotype C1 as containing a risk allele.

Paget disease of bone: mapping of two loci at 5q35-qter and 5q31.

Paget disease of bone is characterized by focal increases of the bone-remodeling process. It is the second most common metabolic bone disease after osteoporosis. Genetic factors play a major role in the etiology of Paget disease of bone, and two loci have been mapped for the disorder: PDB1 and PDB2. The gene(s) causing the typical form of the disorder remains to be characterized. To decipher the molecular basis of Paget disease of bone, we performed genetic linkage analysis in 24 large French Canadian families (479 individuals) in which the disorder was segregating as an autosomal dominant trait. After exclusion of PDB2, a genomewide scan was performed on the three most informative family nuclei. LOD scores >1.0 were observed at seven locations. The 24 families were then used to detect strong evidence for linkage to chromosome 5q35-qter. Under heterogeneity, a maximum LOD score of 8.58 was obtained at D5S2073, at straight theta= .1. The same characteristic haplotype was carried by all patients in eight families, suggesting a founder effect. A recombination event in a key family confined the disease region within a 6-cM interval between D5S469 and the telomere. The 16 other families, with very low conditional probability of linkage to 5q35-qter, were further used, to map a second locus at 5q31. Under heterogeneity, a maximum LOD score of 3.70 was detected at D5S500 with straight theta=.00. Recombination events refined the 5q31 region within 12.2 cM, between D5S642 and D5S1972. These observations demonstrate the mapping of two novel loci for Paget disease of bone and provide further evidence for genetic heterogeneity of this highly prevalent disorder. It is proposed that the 5q35-qter and 5q31 loci be named "PDB3" and "PDB4," respectively.

The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.

OBJECTIVE: To directly ascertain the physiological roles in adipocytes of hormone-sensitive lipase (HSL; E.C. 3.1.1.3), a multifunctional hydrolase that can mediate triacylglycerol cleavage in adipocytes. RESEARCH METHODS AND PROCEDURES: We performed constitutive gene targeting of the mouse HSL gene (Lipe), subsequently studied the adipose tissue phenotype clinically and histologically, and measured lipolysis in isolated adipocytes. RESULTS: Homozygous HSL-/- mice have no detectable HSL peptide or cholesteryl esterase activity in adipose tissue, and heterozygous mice have intermediate levels with respect to wild-type and deficient littermates. HSL-deficient mice have normal body weight but reduced abdominal fat mass compared with normal littermates. Histologically, both white and brown adipose tissues in HSL-/- mice show marked heterogeneity in cell size, with markedly enlarged adipocytes juxtaposed to cells of normal morphology. In isolated HSL-/- adipocytes, lipolysis is not significantly increased by beta3-adrenergic stimulation, but under basal conditions in the absence of added catecholamines, the lipolytic rate of isolated HSL-/- adipocytes is at least as high as that of cells from normal controls. Cold tolerance during a 48-hour period at 4 degrees C was similar in HSL-/- mice and controls. Overnight fasting was well-tolerated clinically by HSL-/- mice, but after fasting, liver triglyceride content was significantly lower in HSL-/- mice compared with wild-type controls. CONCLUSIONS: In isolated fat cells, the lipolytic rate after beta-adrenergic stimulation is mainly dependent on HSL. However, the observation of a normal rate of lipolysis in unstimulated HSL-/- adipocytes suggests that HSL-independent lipolytic pathway(s) exist in fat. Physiologically, HSL deficiency in mice has a modest effect under normal fed conditions and is compatible with normal maintenance of core body temperature during cold stress. However, the lipolytic response to overnight fasting is subnormal.

The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue.

Hormone-sensitive lipase (HSL) mediates triglyceride hydrolysis in adipocytes, in which its expression varies with physiological stress and is controlled posttranslationally and transcriptionally. We sequenced the mouse HSL gene for 8.2 kb upstream of the translation start codon and studied the steady-state HSL mRNA levels in mouse adipose tissue. In 50 clones derived from primer extension and PCR of mouse adipose cDNA, we found five distinct 5' extremities that correspond to distinct exons in genomic DNA. Exon A is located approximately 7 kb 5' to the HSL translation start site. Exons B. C, and D are clustered 1.5-2 kb upstream, and the previously described exon 1 is immediately upstream and contiguous with the previously described HSL translation start site. Exon A is located -7 kb upstream and contains an in-frame methionine codon that could potentially generate another HSL isoform with 43 additional N-terminal residues. cDNA clones containing the newly described exons suggested that each exon has several transcription start sites but that all splice to an acceptor site located 20 nt upstream of the translation initiation codon in exon 1. HSL transcription in mouse adipose tissue originates from multiple sites in the 7-kb region between exon A and exon 1, with peaks at exon C (50-70% of HSL transcripts), exon 1 (5-30%), and exon A (approximately 10%). There are multiple potential transcription factor-binding elements upstream of each exon, suggesting the possibility of differential transcriptional regulation of HSL in different tissues and under various physiologic conditions.

Three-phase bone scintigraphy. Asymmetric patterns in the upper extremities of asymptomatic normals and reflex sympathetic dystrophy patients.

Three-phase Tc-99m MDP scans of 61 patients with asymptomatic upper extremities randomly mixed with 17 studies of patients previously diagnosed with reflex sympathetic dystrophy were blindly interpreted by three observers. Asymmetry in any of the phases was recorded and a final diagnostic impression made. Thirteen of 17 reflux sympathetic dystrophy studies were rated abnormal by at least two observers. Mild to striking asymmetry was occasionally seen in all three phases in asymptomatic upper extremities. Twenty of 61 asymptomatic patients (33%) were rated abnormal by at least one observer, and 5 of 61 studies (8%) were rated abnormal by all observers. Asymmetries in normal patients occurred more commonly in the earlier phases, while asymmetry in the delayed images was mild in all but one. Tightening the criteria to exclude mild asymmetry in delayed images resulted in unacceptably low sensitivity for reflex sympathetic dystrophy (29%). Interobserver variability was most prominent in the flow and immediate images. In the diagnosis of reflex sympathetic dystrophy a greater reliance should be placed on the delayed images, which in themselves have an overall sensitivity of 94%. It is important, however, to recognize that occasional mild and rare moderate asymmetries in even the delayed images of normal individuals result in an overall lower specificity of 77%.

Markers of left ventricular dysfunction induced by exercise, dipyridamole or combined stress on ECG-gated myocardial perfusion scans.

An index of left ventricular contraction can be extracted from the cavitary time-activity curve of electrocardiographic (ECG)-gated myocardial perfusion scans. To assess the induction of stress-induced myocardial depression, we compared contraction indexes derived from immediate poststress and delayed 201Tl images with indexes of ventricular dilation and lung uptake in the prediction of severe coronary artery disease (defined as two or more 90% stenoses). Stress procedures were performed in 93 patients with symptom-limited supine bicycle exercise alone, and in 227 with intravenous dipyridamole, combined where possible with exercise. The immediate and delayed contraction indexes reflected left ventricular dysfunction on ventriculography (P < 0.0001), but additionally the immediate index was reduced (P < 0.0001) in severe coronary disease. Stress-induced hypokinesis was seen frequently after each of the test modes. The relationship with angiographic findings was better defined for indexes of contraction than for lung uptake or ventricular dilation (P < 0.01). The prediction of severe coronary disease was optimized by combining the poststress contraction index and lung uptake. These data support the use of ECG-gated myocardial scans in evaluating the functional consequences of stress/imaging procedures.

Ischemic left ventricular dysfunction assessed on ECG-gated thallium-201 myocardial perfusion images.

Ischemic dysfunction of the left ventricle can be suggested by ancillary data derived from thallium-201 myocardial perfusion images. In this study, qualitative and quantitative assessments of global and segmental contraction derived from ECG-gated left anterior oblique images were analyzed to define more precisely transient ischemic hypokinesis. Immediate (4 mins post stress) and delayed (2 to 4 h) images were compared in 200 patients; 165 had coronary angiography and 35 had a low probability of coronary artery disease based on pretest and test outcome variables. For both immediate and delayed images, a quantitative index of left ventricular contraction (derived from the time-activity curve of the left ventricular cavity and validated in a previous study), correlated well with contrast ventriculography scores. The index derived from the immediate image also was related to the severity/extent of coronary artery lesions and to thallium-201 lung uptake. The ratio of indices (immediate/delayed) was depressed (P less than 0.001) in patients with two or three critically diseased vessels, and reflected the qualitative assessment of stress-induced dysfunction on cinematic images. These data suggest that the quantitative index derived from ECG-gated perfusion scans may be a valuable indicator of stress-induced ventricular contractile dysfunction.

Multimodality imaging of early heterotopic bone formation.

An atypical heterotopic bone formation that was difficult to diagnose presented in a young paraplegic patient as an acute deep vein thrombosis. A number of imaging methods, including contrast venography, ultrasonography, conventional radiography, bone scanning, leukocyte scanning, computed tomography, and magnetic resonance imaging, were used to arrive eventually at the final diagnosis. Early bone scanning remains a sensitive and effective method of diagnosis. Computed tomography can be useful in difficult cases, but the role of other imaging studies appears limited.

Dipyridamole combined with symptom-limited exercise for myocardial perfusion scintigraphy: image characteristics and clinical role.

Although dipyridamole can be used with myocardial scintigraphy to demonstrate reversible perfusion defects, combining exercise with the pharmacologic tool could improve image quality and information yield. The incidence of perfusion defects and the quality of thallium 201 images were reviewed in a series of 820 patients who had been assigned to a specific stress-test mode. Supine bicycle exercise alone was used (group I) where no pharmacologic or physical factors (e.g., beta-blockers, arthritis) limited performance; otherwise, intravenous dipyridamole was followed by symptom-limited exercise (group II). Angiographic correlation was available in 57 patients in group I, and in 158 in group II; of these, 109 performed significant exercise (greater than or equal to 3 min at increasing workloads) following dipyridamole (group IIA), whereas in 49 (group IIB) the exercise phase following dipyridamole was truncated. All test-mode groups were similar with respect to the incidence of ST segment depression during testing, patient throughput, and the sensitivity of perfusion defects. Chest pain and reversible defects were induced more frequently in group II than in group I. In group IIA, splanchnic background activity was lower (P less than 0.001) than in group IIB, and the false-positive rate tended to be lower. Thus, combining exercise with dipyridamole in patients with non-cardiac limitations to exercise enabled the achievement of optimal results for perfusion scintigraphy.

Cerebral perfusion imaging with technetium-99m HM-PAO in brain death and severe central nervous system injury.

We performed 38 cerebral perfusion studies in 33 patients with brain death or with severe central nervous system injury using technetium-99m hexamethyl-propyleneamine oxime [( 99mTc]HM-PAO). Uptake by the cerebrum and/or cerebellium was present in all patients who were not clinically brain dead (ten studies) although the study was often abnormal. In those patients who were brain dead, 16/17 studies demonstrated no uptake in either the cerebrum or cerebellum. In patients suspected of brain death, but who had conditions interfering with the diagnosis the test demonstrated no uptake in 9/11 studies, confirming brain death. A radionuclide angiogram (RNA) of the head was also performed in 33/38 studies and showed complete agreement with the [99mTc]HM-PAO uptake, except in one case. We conclude that cerebral perfusion imaging with [99mTc]HM-PAO is a simple, noninvasive and reliable test to confirm brain death. By comparison with conventional technetium agents, [99mTc]HM-PAO is not dependent on the quality of the bolus injection, is easier to interpret and allows evaluation of posterior fossa blood flow.

Estimation of left ventricular function on ECG-gated thallium-201 myocardial perfusion images.

ECG-gated thallium-201 perfusion images (GT) may provide information concerning left ventricular contraction. To assess the objectivity of this information, qualitative and quantitative evaluations of left anterior oblique GT images were performed in 600 patients and compared with contrast ventriculography (n = 180) and gated blood pool scintigraphy (n = 60). Cinematic playback of GT allowed qualitative rating of ventricular performance in 98% of studies. Abnormal GT (focal or global hypokinesis) was found in 41% of cases and could be related to myocardial infarction, nonischemic cardiomyopathy or left bundle branch block. Blinded readings showed reasonable interobserver agreement and correlation with blood pool scintigraphy regarding segmental and overall left ventricular function; decreased tracer uptake in an abnormally perfused segment presented only occasional difficulty. A quantitative index was derived by relating the increment in left ventricular cavity activity during contraction to myocardial count density; results were expressed as a planar ejection fraction (PEF). PEF correlated well with ejection fraction on blood pool scintigraphy (r = 0.83, P less than 0.001) and with contractility scores derived from ventriculography (P less than 0.001) and seemed suited for identification of hypokinetic ventricles. Thus, GT allows objective estimation of left ventricular function as an addendum to myocardial perfusion imaging.