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Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a robust genetic influence. The norepinephrine transporter (NET) is of particular interest as it is one of the main targets in treatment of the disorder. As ADHD is a complex and polygenetic condition, the possible regulation by epigenetic processes has received increased attention. We sought to determine possible differences in NET promoter DNA methylation between patients with ADHD and healthy controls. DNA methylation levels in the promoter region of the NET were determined in 23 adult patients with ADHD and 23 healthy controls. A subgroup of 18 patients with ADHD and 18 healthy controls underwent positron emission tomography (PET) with the radioligand (S,S)-[ 18 F]FMeNER-D 2 to quantify the NET in several brain areas in vivo. Analyses revealed significant differences in NET methylation levels at several cytosine-phosphate-guanine (CpG) sites between groups. A defined segment of the NET promoter ("region 1") was hypermethylated in patients in comparison with controls. In ADHD patients, a negative correlation between methylation of a CpG site in this region and NET distribution in the thalamus, locus coeruleus, and the raphe nuclei was detected. Furthermore, methylation of several sites in region 1 was negatively associated with the severity of hyperactivity-impulsivity symptoms. Our results point to an epigenetic dysregulation in ADHD, possibly due to a compensatory mechanisms or additional factors involved in transcriptional processing.

Background: Several MR-based attenuation correction (AC) approaches were developed to conquer the challenging AC in hybrid PET/MR imaging. These AC methods are commonly evaluated on standardized uptake values or tissue concentration. However, in neurotransmitter system studies absolute quantification is more favorable due to its accuracy. Therefore, our aim was to investigate the accuracy of segmentation- and atlas-based MR AC approaches on serotonin transporter (SERT) distribution volumes and occupancy after a drug challenge., Methods: 18 healthy subjects (7 male) underwent two [ 11 C]DASB PET/MRI measurements in a double-blinded, placebo controlled, cross-over design. After 70 min the selective serotonin reuptake inhibitor (SSRI) citalopram or a placebo was infused. The parameters total and specific volume of distribution (V T , V S = BP P ) and occupancy were quantified. All subjects underwent a low-dose CT scan as reference AC method. Besides the standard AC approaches DIXON and UTE, a T1-weighted structural image was recorded to estimate a pseudo-CT based on an MR/CT database (pseudoCT). Another evaluated AC approach superimposed a bone model on AC DIXON. Lastly, an approach optimizing the segmentation of UTE images was analyzed (RESOLUTE). PET emission data were reconstructed with all 6 AC methods. The accuracy of the AC approaches was evaluated on a region of interest-basis for the parameters V T , BP P , and occupancy with respect to the results of AC CT., Results: Variations for V T and BP P were found with all AC methods with bias ranging from -15 to 17%. The smallest relative errors for all regions were found with AC pseudoCT (<|5%|). Although the bias between BP P SSRI and BP P placebo varied markedly with AC DIXON (<|12%|) and AC UTE (<|9%|), a high correlation to AC CT was obtained ( r 2 ∼1). The relative difference of the occupancy for all tested AC methods was small for SERT high binding regions (<|4%|)., Conclusion: The high correlation might offer a rescaling from the biased parameters V T and BP P to the true values. Overall, the pseudoCT approach yielded smallest errors and the best agreement with AC CT. For SERT occupancy, all AC methods showed little bias in high binding regions, indicating that errors may cancel out in longitudinal assessments., (Copyright © 2019 Rischka, Gryglewski, Berroterán-Infante, Rausch, James, Klöbl, Sigurdardottir, Hartenbach, Hahn, Wadsak, Mitterhauser, Beyer, Kasper, Prayer, Hacker and Lanzenberger.)

Introduction: In-vivo quantification of the serotonin transporter (SERT) guided our understanding of many neuropsychiatric disorders. A recently introduced bolus plus constant infusion protocol has been shown to allow the reliable determination of SERT binding with reduced scan time. In this work, the outcomes of two methods, a bolus injection paradigm on a GE PET camera, and a bolus plus infusion paradigm on a combined Siemens PET/MR camera were compared. Methods: A total of seven healthy subjects underwent paired PET and paired PET/MR scans each with intravenous double-blind application of 7.5 mg citalopram or saline in a randomized cross-over study design. While PET scans were performed according to standard protocols and non-displaceable binding potentials (BP ND ) were calculated using the multi-linear reference tissue model, during PET/MR measurements [ 11 C]DASB was applied as bolus plus constant infusion, and BP ND was calculated using the steady state method and data acquired at tracer equilibrium. Occupancies were calculated as the relative decrease in BP ND between saline and citalopram scans. Results: During placebo scans, a mean difference in BP ND of -0.08 (-11.71%) across all ROIs was found between methods. PET/MR scans resulted in higher BP ND estimates than PET scans in all ROIs except the midbrain. A mean difference of -0.19 (-109.40%) across all ROIs between methods was observed for citalopram scans. PET/MR scans resulted in higher BP ND estimates than PET scans in all ROIs. For occupancy, a mean difference of 23.12% (21.91%) was observed across all ROIs. PET/MR scans resulted in lower occupancy compared to PET scans in all ROIs except the temporal cortex. While for placebo, BP ND of high-binding regions (thalamus and striatum) exhibited moderate reliability (ICC = 0.66), during citalopram scans ICC decreased (0.36-0.46). However, reliability for occupancy remained high (0.57-0.82). Conclusion: Here, we demonstrated the feasibility of reliable and non-invasive SERT quantification using a [ 11 C]DASB bolus plus constant infusion protocol at a hybrid PET/MR scanner, which might facilitate future pharmacological imaging studies. Highest agreement with established methods for quantification of occupancy and SERT BP ND at baseline was observed in subcortical high-binding regions.

Pharmacological imaging of the effects of selective serotonin reuptake inhibitors (SSRI) may aid the clarification of their mechanism of action and influence treatment of highly prevalent neuropsychiatric conditions if the detected effects could be related to patient outcomes. In a randomized double-blind design, 38 healthy participants received a constant infusion of 8 mg citalopram or saline during either their first or second of two PET/MR scans. Resting-state functional MRI (fMRI) was acquired simultaneously with PET data on the binding of serotonin transporters (5-HTT) using [ 11 C]DASB. Three different approaches for modeling of pharmacological fMRI response were tested separately. These relied on the use of regressors corresponding to (1) the drug infusion paradigm, (2) time courses of citalopram plasma concentrations and (3) changes in 5-HTT binding measured in each individual, respectively. Furthermore, the replication of results of a widely used model-free analysis method was attempted which assesses the deviation of signal in discrete time bins of fMRI data acquired after start of drug infusion. Following drug challenge, average 5-HTT occupancy was 69±7% and peak citalopram plasma levels were 111.8 ± 21.1 ng/ml. None of the applied methods could detect significant differences in the pharmacological response between SSRI and placebo scans. The failed replication of SSRI effects reported in the literature despite a threefold larger sample size highlights the importance of appropriate correction for family-wise error in order to avoid spurious results in pharmacological imaging. This calls for the development of analysis methods which take regional specialization and the dynamics of brain activity into account., (Copyright © 2019 Elsevier B.V. and ECNP. All rights reserved.)

Background: Electroconvulsive therapy (ECT) constitutes one of the most effective antidepressant treatment strategies in major depression (MDD). Despite its common use and uncontested efficacy, its mechanism of action is still insufficiently understood. Previously, we showed that ECT is accompanied by a global decrease of serotonin-1A receptors in MDD; however, further studies to investigate the involvement of the serotonergic system in the mechanism of action of ECT are warranted. The monoamine oxidase A (MAO-A) represents an important target for antidepressant treatments and was found to be increased in MDD. Here, we investigated whether ECT impacts on MAO-A levels in treatment-resistant patients (TRD)., Methods: 16 TRD patients (12 female, age 45.94 ± 9.68 years, HAMD 25.12 ± 3.16) with unipolar depression according to DSM-IV were scanned twice before (PET1 and PET2, to assess test-retest variability under constant psychopharmacotherapy) and once after (PET3) completing a minimum of eight unilateral ECT sessions using positron emission tomography and the radioligand [ 11 C]harmine to assess cerebral MAO-A distribution volumes (V T ). Age- and sex-matched healthy subjects (HC) were measured once., Results: Response rate to ECT was 87.5%. MAO-A V T was found to be significantly reduced after ECT in TRD patients (-3.8%) when assessed in 27 a priori defined ROIs (p < 0.001). Test-retest variability between PET1 and PET2 was 3.1%. MAO-A V T did not significantly differ between TRD patients and HC at baseline., Conclusions: The small effect size of the significant reduction of MAO-A V T after ECT in the range of test-retest variability does not support the hypothesis of a clinically relevant mechanism of action of ECT based on MAO-A. Furthermore, in contrast to studies reporting elevated MAO-A V T in unmedicated depressed patients, MAO-A levels were found to be similar in TRD patients and HC which might be attributed to the continuous antidepressant pharmacotherapy in the present sample., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Alterations of the 5-HT 1A receptor and BDNF have consistently been associated with affective disorders. Two functional single nucleotide polymorphisms (SNPs), rs6295 of the serotonin 1A receptor gene (HTR1A) and rs6265 of brain-derived neurotrophic factor gene (BDNF), may impact transcriptional regulation and expression of the 5-HT 1A receptor. Here we investigated interaction effects of rs6295 and rs6265 on 5-HT 1A receptor binding. Forty-six healthy subjects were scanned with PET using the radioligand [carbonyl- 11 C]WAY-100635. Genotyping was performed for rs6265 and rs6295. Subjects showing a genotype with at least three risk alleles (G of rs6295 or A of rs6265) were compared to control genotypes. Cortical surface binding potential (BP ND ) was computed for 32 cortical regions of interest (ROI). Mixed model was applied to study main and interaction effects of ROI and genotype. ANOVA was used for post hoc analyses. Individuals with the risk genotypes exhibited an increase in 5-HT 1A receptor binding by an average of 17% (mean BP ND 3.56 ± 0.74 vs. 2.96 ± 0.88). Mixed model produced an interaction effect of ROI and genotype on BP ND and differences could be demonstrated in 10 ROI post hoc. The combination of disadvantageous allelic expression of rs6295 and rs6265 may result in a 5-HT 1A receptor profile comparable to affective disorders as increased 5-HT 1A receptor binding is a well published phenotype of depression. Thus, epistasis between BDNF and HTR1A may contribute to the multifactorial risk for affective disorders and our results strongly advocate further research on this genetic signature in affective disorders.

Parcellation of distinct areas in the cerebral cortex has a long history in neuroscience and is of great value for the study of brain function, specialization, and alterations in neuropsychiatric disorders. Analysis of cytoarchitectonical features has revealed their close association with molecular profiles based on protein density. This provides a rationale for the use of in vivo molecular imaging data for parcellation of the cortex with the advantage of whole-brain coverage. In the current work, parcellation was based on expression of key players of the serotonin neurotransmitter system. Positron emission tomography was carried out for the quantification of serotonin 1A (5-HT1A, n = 30) and 5-HT2A receptors (n = 22), the serotonin-degrading enzyme monoamine oxidase A (MAO-A, n = 32) and the serotonin transporter (5-HTT, n = 24) in healthy participants. Cortical protein distribution maps were obtained using surface-based quantification. Based on k-means clustering, silhouette criterion and bootstrapping, five distinct clusters were identified as the optimal solution. The defined clusters proved of high explanatory value for the effects of psychotropic drugs acting on the serotonin system, such as antidepressants and psychedelics. Therefore, the proposed method constitutes a sensible approach towards integration of multimodal imaging data for research and development in neuropharmacology and psychiatry.

Increased cerebral monoamine oxidase A (MAO-A) levels have been shown in non-seasonal depression using positron emission tomography (PET). Seasonal affective disorder (SAD) is a sub-form of major depressive disorder and is typically treated with bright light therapy (BLT). The serotonergic system is affected by season and light. Hence, this study aims to assess the relevance of brain MAO-A levels to the pathophysiology and treatment of SAD. Changes to cerebral MAO-A distribution (1) in SAD in comparison to healthy controls (HC), (2) after treatment with BLT and (3) between the seasons, were investigated in 24 patients with SAD and 27 HC using [ 11 C]harmine PET. PET scans were performed in fall/winter before and after 3 weeks of placebo-controlled BLT, as well as in spring/summer. Cerebral MAO-A distribution volume (V T , an index of MAO-A density) did not differ between patients and HC at any of the three time-points. However, MAO-A V T decreased from fall/winter to spring/summer in the HC group (F 1, 187.84  = 4.79, p < 0.050), while SAD showed no change. In addition, BLT, but not placebo, resulted in a significant reduction in MAO-A V T (F 1, 208.92  = 25.96, p < 0.001). This is the first study to demonstrate an influence of BLT on human cerebral MAO-A levels in vivo. Furthermore, we show that SAD may lack seasonal dynamics in brain MAO-A levels. The lack of a cross-sectional difference between patients and HC, in contrast to studies in non-seasonal depression, may be due to the milder symptoms typically shown by patients with SAD.

The quantification of big pools of diverse molecules provides important insights on brain function, but is often restricted to a limited number of observations, which impairs integration with other modalities. To resolve this issue, a method allowing for the prediction of mRNA expression in the entire brain based on microarray data provided in the Allen Human Brain Atlas was developed. Microarray data of 3702 samples from 6 brain donors was registered to MNI and cortical surface space using FreeSurfer. For each of 18,686 genes, spatial dependence of transcription was assessed using variogram modelling. Variogram models were employed in Gaussian process regression to calculate best linear unbiased predictions for gene expression at all locations represented in well-established imaging atlases for cortex, subcortical structures and cerebellum. For validation, predicted whole-brain transcription of the HTR1A gene was correlated with [carbonyl- 11 C]WAY-100635 positron emission tomography data collected from 30 healthy subjects. Prediction results showed minimal bias ranging within ±0.016 (cortical surface), ±0.12 (subcortical regions) and ±0.14 (cerebellum) in units of log2 expression intensity for all genes. Across genes, the correlation of predicted and observed mRNA expression in leave-one-out cross-validation correlated with the strength of spatial dependence (cortical surface: r = 0.91, subcortical regions: r = 0.85, cerebellum: r = 0.84). 816 out of 18,686 genes exhibited a high spatial dependence accounting for more than 50% of variance in the difference of gene expression on the cortical surface. In subcortical regions and cerebellum, different sets of genes were implicated by high spatially structured variability. For the serotonin 1A receptor, correlation between PET binding potentials and predicted comprehensive mRNA expression was markedly higher (Spearman ρ = 0.72 for cortical surface, ρ = 0.84 for subcortical regions) than correlation of PET and discrete samples only (ρ = 0.55 and ρ = 0.63, respectively). Prediction of mRNA expression in the entire human brain allows for intuitive visualization of gene transcription and seamless integration in multimodal analysis without bias arising from non-uniform distribution of available samples. Extension of this methodology promises to facilitate translation of omics research and enable investigation of human brain function at a systems level., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Comprehensive description of ketamine's molecular binding profile becomes increasingly pressing as use in real-life patient cohorts widens. Animal studies attribute a significant role in the substance's antidepressant effects to the serotonergic system. The serotonin transporter is a highly relevant target in this context, because it is central to depressive pathophysiology and treatment. This is, to our knowledge, the first study investigating ketamine's serotonin transporter binding in vivo in humans., Methods: Twelve healthy subjects were assessed twice using [11C]DASB positron emission tomography. A total of 0.50 mg/kg bodyweight ketamine was administered once i.v. prior to the second positron emission tomography scan. Ketamine plasma levels were determined during positron emission tomography. Serotonin transporter nondisplaceable binding potential was computed using a reference region model, and occupancy was calculated for 4 serotonin transporter-rich regions (caudate, putamen, thalamus, midbrain) and a whole-brain region of interest., Results: After administration of the routine antidepressant dose, ketamine showed <10% occupancy of the serotonin transporter, which is within the test-retest variability of [11C]DASB. A positive correlation between ketamine plasma levels and occupancy was shown., Conclusions: Measurable occupancy of the serotonin transporter was not detectable after administration of an antidepressant dose of ketamine. This might suggest that ketamine binding of the serotonin transporter is unlikely to be a primary antidepressant mechanism at routine antidepressant doses, as substances that facilitate antidepressant effects via serotonin transporter binding (e.g., selective serotonin reuptake inhibitors) show 70% to 80% occupancy. Administration of high-dose ketamine is widening. Based on the positive relationship we find between ketamine plasma levels and occupancy, there is a need for investigation of ketamine's serotonin transporter binding at higher doses., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

Major depressive disorder (MDD) is the most common neuropsychiatric disease and despite extensive research, its genetic substrate is still not sufficiently understood. The common polymorphism rs6295 of the serotonin-1A receptor gene (HTR1A) is affecting the transcriptional regulation of the 5-HT 1A receptor and has been closely linked to MDD. Here, we used positron emission tomography (PET) exploiting advances in data mining and statistics by using machine learning in 62 healthy subjects and 19 patients with MDD, which were scanned with PET using the radioligand [carbonyl- 11 C]WAY-100635. All the subjects were genotyped for rs6295 and genotype was grouped in GG vs C allele carriers. Mixed model was applied in a ROI-based (region of interest) approach. ROI binding potential (BP ND ) was divided by dorsal raphe BP ND as a specific measure to highlight rs6295 effects (BP Div ). Mixed model produced an interaction effect of ROI and genotype in the patients' group but no effects in healthy controls. Differences of BP Div was demonstrated in seven ROIs; parahippocampus, hippocampus, fusiform gyrus, gyrus rectus, supplementary motor area, inferior frontal occipital gyrus and lingual gyrus. For classification of genotype, 'RandomForest' and Support Vector Machines were used, however, no model with sufficient predictive capability could be computed. Our results are in line with preclinical data, mouse model knockout studies as well as previous clinical analyses, demonstrating the two-pronged effect of the G allele on 5-HT 1A BP ND for, we believe, the first time. Future endeavors should address epigenetic effects and allosteric heteroreceptor complexes. Replication in larger samples of MDD patients is necessary to substantiate our findings.

Introduction: In-vivo quantification of serotonin transporters (SERT) in human brain has been a mainstay of molecular imaging in the field of neuropsychiatric disorders and helped to explore the underpinnings of several medical conditions, therapeutic and environmental influences. The emergence of PET/MR hybrid systems and the heterogeneity of SERT binding call for the development of efficient methods making the investigation of larger or vulnerable populations with limited scanner time and simultaneous changes in molecular and functional measures possible. We propose [ 11 C]DASB bolus plus constant infusion for these applications and validate it against standard analyses of dynamic PET data., Methods: [ 11 C]DASB bolus/infusion optimization was performed on data acquired after [ 11 C]DASB bolus in 8 healthy subjects. Subsequently, 16 subjects underwent one scan using [ 11 C]DASB bolus plus constant infusion with K bol 160-179min and one scan after [ 11 C]DASB bolus for inter-method reliability analysis. Arterial blood sampling and metabolite analysis were performed for all scans. Distribution volumes (V T ) were obtained using Logan plots for bolus scans and ratios between tissue and plasma parent activity for bolus plus infusion scans for different time spans of the scan (V T-70 for 60-70min after start of tracer infusion, V T-90 for 75-90min, V T-120 for 100-120min) in 9 subjects. Omitting blood data, binding potentials (BP ND ) obtained using multilinear reference tissue modeling (MRTM2) and cerebellar gray matter as reference region were compared in 11 subjects., Results: A K bol of 160min was observed to be optimal for rapid equilibration in thalamus and striatum. V T-70 showed good intraclass correlation coefficients (ICCs) of 0.61-0.70 for thalamus, striatal regions and olfactory cortex with bias ≤5.1% compared to bolus scans. ICCs increased to 0.72-0.78 for V T-90 and 0.77-0.93 for V T-120 in these regions. BP ND-90 had negligible bias ≤2.5%, low variability ≤7.9% and ICCs of 0.74-0.87; BP ND-120 had ICCs of 0.73-0.90. Low-binding cortical regions and cerebellar gray matter showed a positive bias of ~8% and ICCs 0.57-0.68 at V T-90 . Cortical BP ND suffered from high variability and bias, best results were obtained for olfactory cortex and anterior cingulate cortex with ICC=0.74-0.75 for BP ND-90 . High-density regions amygdala and midbrain had a negative bias of -5.5% and -22.5% at V T-90 with ICC 0.70 and 0.63, respectively., Conclusions: We have optimized the equilibrium method with [ 11 C]DASB bolus plus constant infusion and demonstrated good inter-method reliability with accepted standard methods and for SERT quantification using both V T and BP ND in a range of different brain regions. With as little as 10-15min of scanning valid estimates of SERT V T and BP ND in thalamus, amygdala, striatal and high-binding cortical regions could be obtained. Blood sampling seems vital for valid quantification of SERT in low-binding cortical regions. These methods allow the investigation of up to three subjects with a single radiosynthesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aggregation of hyperphosphorylated tau is a major hallmark of many neurodegenerative diseases, including Alzheimer disease (AD). In vivo imaging with PET may offer important insights into pathophysiologic mechanisms, diagnosis, and disease progression. We describe different strategies for quantification of 18 F-AV-1451 (T807) tau binding, including models with blood sampling and noninvasive alternatives. Methods: Fifteen subjects (4 controls, 6 AD, 3 progressive supranuclear palsy, 2 cortico basal syndrome) underwent 180-min PET with 18 F-AV-1451 and arterial blood sampling. Modeling with arterial input functions included 1-, 2-, and 3-tissue-compartment models and the Logan plot. Using the cerebellum as reference region, we applied the simplified reference tissue model 2 and Logan reference plot. Finally, simplified outcome measures were calculated as ratio, with reference to cerebellar concentrations (SUV ratio [SUVR]) and SUVs. Results: Tissue compartment models were not able to describe the kinetics of 18 F-AV-1451, with poor fits in 33%-53% of cortical regions and 80% in subcortical areas. In contrast, the Logan plot showed excellent fits and parameter variance (total volume of distribution SE < 5%). Compared with the 180-min arterial-based Logan model, strong agreement was obtained for the Logan reference plot also for a reduced scan time of 100 min ( R 2 = 0.91) and SUVR 100-120 min ( R 2 = 0.94), with 80-100 min already representing a reasonable compromise between duration and accuracy ( R 2 = 0.93). Time-activity curves and kinetic parameters were equal for cortical regions and the cerebellum in control subjects but different in the putamen. Cerebellar total volumes of distribution were higher in controls than patients. For these methods, increased cortical binding was observed for AD patients and to some extent for cortico basal syndrome, but not progressive supranuclear palsy. Conclusion: The Logan plot provided the best estimate of tau binding using arterial input functions. Assuming that the cerebellum is a valid reference region, simplified methods seem to provide robust alternatives for quantification, such as the Logan reference plot with 100-min scan time. Furthermore, SUVRs between target and cerebellar activities obtained from an 80- to 100-min static scan offer promising potential for clinical routine application., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

Selective serotonin reuptake inhibitors (SSRIs) modulate serotonergic neurotransmission by blocking reuptake of serotonin from the extracellular space. Up to now, it remains unclear how SSRIs achieve their antidepressant effect. However, task-based and resting state functional magnetic resonance imaging studies, have demonstrated connectivity changes between brain regions. Here, we use positron emission tomography (PET) to quantify SSRI's main target, the serotonin transporter (SERT), and assess treatment-induced molecular changes in the interregional relation of SERT binding potential (BP ND ). Nineteen out-patients with major depressive disorder (MDD) and 19 healthy controls (HC) were included in this study. Patients underwent three PET measurements with the radioligand [ 11 C]DASB: (1) at baseline, (2) after a first SSRI dose; and (3) following at least 3 weeks of daily intake. Controls were measured once with PET. Correlation analyses were restricted to brain regions repeatedly implicated in MDD pathophysiology. After 3 weeks of daily SSRI administration a significant increase in SERT BP ND correlations of anterior cingulate cortex and insula with the amygdala, midbrain, hippocampus, pallidum and putamen ( p < 0.05; false discovery rate, FDR corrected) was revealed. No significant differences were found when comparing MDD patients and HC at baseline. These findings are in line with the clinical observation that treatment response to SSRIs is often achieved only after a latency of several weeks. The elevated associations in interregional SERT associations may be more closely connected to clinical outcomes than regional SERT occupancy measures and could reflect a change in the regional interaction of serotonergic neurotransmission during antidepressant treatment.

Altered serotonergic neurotransmission has been found to cause impulsive and aggressive behavior, as well as increased motor activity, all exemplifying key symptoms of ADHD. The main objectives of this positron emission tomography (PET) study were to investigate the serotonin transporter binding potential (SERT BP ND ) in patients with ADHD and to assess associations of SERT BP ND between the brain regions. 25 medication-free patients with ADHD (age ± SD; 32.39 ± 10.15; 10 females) without any psychiatric comorbidity and 25 age and sex matched healthy control subjects (33.74 ± 10.20) were measured once with PET and the highly selective and specific radioligand [ 11 C]DASB. SERT BP ND maps in nine a priori defined ROIs exhibiting high SERT binding were compared between groups by means of a linear mixed model. Finally, adopted from structural and functional connectivity analyses, we performed correlational analyses using regional SERT binding potentials to examine molecular interregional associations between all selected ROIs. We observed significant differences in the interregional correlations between the precuneus and the hippocampus in patients with ADHD compared to healthy controls, using SERT BP ND of the investigated ROIs (P < 0.05; Bonferroni corrected). When correlating SERT BP ND and age in the ADHD and the healthy control group, we confirmed an age-related decline in brain SERT binding in the thalamus and insula (R 2  = 0.284, R 2  = 0.167, Ps < 0.05; Bonferroni corrected). The results show significantly different interregional molecular associations of the SERT expression for the precuneus with hippocampus in patients with ADHD, indicating presumably altered functional coupling. Altered interregional coupling between brain regions might be a sensitive approach to demonstrate functional and molecular alterations in psychiatric conditions. Hum Brain Mapp 38:792-802, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Regional differences in posttranscriptional mechanisms may influence in vivo protein densities. The association of positron emission tomography (PET) imaging data from 112 healthy controls and gene expression values from the Allen Human Brain Atlas, based on post-mortem brains, was investigated for key serotonergic proteins. PET binding values and gene expression intensities were correlated for the main inhibitory (5-HT1A) and excitatory (5-HT2A) serotonin receptor, the serotonin transporter (SERT) as well as monoamine oxidase-A (MAO-A), using Spearman's correlation coefficients (rs) in a voxel-wise and region-wise analysis. Correlations indicated a strong linear relationship between gene and protein expression for both the 5-HT1A (voxel-wise rs = 0.71; region-wise rs = 0.93) and the 5-HT2A receptor (rs = 0.66; 0.75), but only a weak association for MAO-A (rs = 0.26; 0.66) and no clear correlation for SERT (rs = 0.17; 0.29). Additionally, region-wise correlations were performed using mRNA expression from the HBT, yielding comparable results (5-HT1Ars = 0.82; 5-HT2Ars = 0.88; MAO-A rs = 0.50; SERT rs = -0.01). The SERT and MAO-A appear to be regulated in a region-specific manner across the whole brain. In contrast, the serotonin-1A and -2A receptors are presumably targeted by common posttranscriptional processes similar in all brain areas suggesting the applicability of mRNA expression as surrogate parameter for density of these proteins., (© The Author 2016. Published by Oxford University Press.)

The investigation of cerebral metabolic rate of glucose (CMRGlu) at baseline and during specific tasks previously required separate scans with the drawback of high intrasubject variability. We aimed to validate a novel approach to assessing baseline glucose metabolism and task-specific changes in a single measurement with a constant infusion of 18 F-FDG., Methods: Fifteen healthy subjects underwent two PET measurements with arterial blood sampling. As a reference, baseline CMRGlu was quantified from a 60-min scan after 18 F-FDG bolus application using the Patlak plot (eyes closed). For the other scan, a constant radioligand infusion was applied for 95 min, during which the subjects opened their eyes at 10-20 min and 60-70 min and tapped their right thumb to their fingers at 35-45 min and 85-95 min. The constant-infusion scan was quantified in two steps. First, the general linear model was used to fit regional time-activity curves with regressors for baseline metabolism, task-specific changes for the eyes-open and finger-tapping conditions, and movement parameters. Second, the Patlak plot was used for quantification of CMRGlu. Multiplication of the baseline regressor by β-values from the general linear model yielded regionally specific time-activity curves for baseline metabolism. Further, task-specific changes in metabolism are directly proportional to changes in the slope of the time-activity curve and hence to changes in CMRGlu., Results: Baseline CMRGlu from the constant-infusion scan matched that from the bolus application (test-retest variability, 1.1% ± 24.7%), which was not the case for a previously suggested approach (variability, -39.9% ± 25.2%, P < 0.001). Task-specific CMRGlu increased in the primary visual and motor cortices for eyes open and finger tapping, respectively (P < 0.05, familywise error-corrected), with absolute changes of up to 2.1 μmol/100 g/min and 6.3% relative to baseline. For eyes open, a decreased CMRGlu was observed in default-mode regions (P < 0.05, familywise error-corrected). CMRGlu quantified with venous blood samples (n = 6) showed excellent agreement with results obtained from arterial samples (r > 0.99)., Conclusion: Baseline glucose metabolism and task-specific changes can be quantified in a single measurement with constant infusion of 18 F-FDG and venous blood sampling. The high sensitivity and regional specificity of the approach offer novel possibilities for functional and multimodal brain imaging., (© 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disorder with a strong genetic component. The norepinephrine transporter (NET) is a key target for ADHD treatment and the NET gene has been of high interest as a possible modulator of ADHD pathophysiology. Therefore, we conducted an imaging genetics study to examine possible effects of single nucleotide polymorphisms (SNPs) within the NET gene on NET nondisplaceable binding potential (BPND ) in patients with ADHD and healthy controls (HCs). Twenty adult patients with ADHD and 20 HCs underwent (S,S)-[18F]FMeNER-D2 positron emission tomography (PET) and were genotyped on a MassARRAY MALDI-TOF platform using the Sequenom iPLEX assay. Linear mixed models analyses revealed a genotype-dependent difference in NET BPND between groups in the thalamus and cerebellum. In the thalamus, a functional promoter SNP (-3081 A/T) and a 5'-untranslated region (5'UTR) SNP (-182 T/C), showed higher binding in ADHD patients compared to HCs depending on the major allele. Furthermore, we detected an effect of genotype in HCs, with major allele carriers having lower binding. In contrast, for two 3'UTR SNPs (*269 T/C, *417 A/T), ADHD subjects had lower binding in the cerebellum compared to HCs depending on the major allele. Additionally, symptoms of hyperactivity and impulsivity correlated with NET BPND in the cerebellum depending on genotype. Symptoms correlated positively with cerebellar NET BPND for the major allele, while symptoms correlated negatively to NET BPND in minor allele carriers. Our findings support the role of genetic influence of the NE system on NET binding to be pertubated in ADHD., (© 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.)

In 2009, the General Medical Council UK (GMC) published its updated guidance on medical education for the UK medical schools - Tomorrow's Doctors 2009. The Council recommended that the UK medical schools introduce, for the first time, a clinical placement in which a senior medical student, "assisting a junior doctor and under supervision, undertakes most of the duties of an F1 doctor". In the UK, an F1 doctor is a postgraduation year 1 (PGY1) doctor. This new kind of placement was called a student assistantship. The recommendation was considered necessary because conventional UK clinical placements rarely provided medical students with opportunities to take responsibility for patients - even under supervision. This is in spite of good evidence that higher levels of learning, and the acquisition of essential clinical and nontechnical skills, depend on students participating in health care delivery and gradually assuming responsibility under supervision. This review discusses the gap between student and doctor, and the impact of the student assistantship policy. Early evaluation indicates substantial variation in the clarity of purpose, setting, length, and scope of existing assistantships. In particular, few models are explicit on the most critical issue: exactly how the student participates in care and how supervision is deployed to optimize learning and patient safety. Surveys indicate that these issues are central to students' perceptions of the assistantship. They know when they have experienced real responsibility and when they have not. This lack of clarity and variation has limited the impact of student assistantships. We also consider other important approaches to bridging the gap between student and doctor. These include supporting the development of the student as a whole person, commissioning and developing the right supervision, student-aligned curricula, and challenging the risk assumptions of health care providers.

Background: Lactose intolerance (LI) is a common medical problem with limited treatment options. The primary symptoms are abdominal pain, diarrhea, bloating, flatulence, and cramping. Limiting dairy foods to reduce symptoms contributes to low calcium intake and the risk for chronic disease. Adaptation of the colon bacteria to effectively metabolize lactose is a novel and potentially useful approach to improve lactose digestion and tolerance. RP-G28 is novel galacto-oligosaccharide (GOS) being investigated to improve lactose digestion and the symptoms of lactose intolerance in affected patients., Methods: A randomized, double-blind, parallel group, placebo-controlled study was conducted at 2 sites in the United States. RP-G28 or placebo was administered to 85 patients with LI for 35 days. Post-treatment, subjects reintroduced dairy into their daily diets and were followed for 30 additional days to evaluate lactose digestion as measured by hydrogen production and symptom improvements via a patient-reported symptom assessment instrument., Results: Lactose digestion and symptoms of LI trended toward improvement on RP-G28 at the end of treatment and 30 days post-treatment. A reduction in abdominal pain was also demonstrated in the study results. Fifty percent of RP-G28 subjects with abdominal pain at baseline reported no abdominal pain at the end of treatment and 30 days post treatment (p = 0.0190). RP-G28 subjects were also six times more likely to claim lactose tolerance post-treatment once dairy foods had been re-introduced into their diets (p = 0.0389)., Conclusions: Efficacy trends and favorable safety/tolerability findings suggest that RP-G28 appears to be a potentially useful approach for improving lactose digestion and LI symptoms. The concurrent reduction in abdominal pain and improved overall tolerance could be a meaningful benefit to lactose intolerant individuals.

In many organisms the expression levels of each gene are controlled by the activation levels of known "Transcription Factors" (TF). A problem of considerable interest is that of estimating the "Transcription Regulation Networks" (TRN) relating the TFs and genes. While the expression levels of genes can be observed, the activation levels of the corresponding TFs are usually unknown, greatly increasing the difficulty of the problem. Based on previous experimental work, it is often the case that partial information about the TRN is available. For example, certain TFs may be known to regulate a given gene or in other cases a connection may be predicted with a certain probability. In general, the biology of the problem indicates there will be very few connections between TFs and genes. Several methods have been proposed for estimating TRNs. However, they all suffer from problems such as unrealistic assumptions about prior knowledge of the network structure or computational limitations. We propose a new approach that can directly utilize prior information about the network structure in conjunction with observed gene expression data to estimate the TRN. Our approach uses L(1) penalties on the network to ensure a sparse structure. This has the advantage of being computationally efficient as well as making many fewer assumptions about the network structure. We use our methodology to construct the TRN for E. coli and show that the estimate is biologically sensible and compares favorably with previous estimates.

Phenotypes are complex, and difficult to quantify in a high-throughput fashion. The lack of comprehensive phenotype data can prevent or distort genotype-phenotype mapping. Here, we describe "PhenoProfiler," a computational method that enables in silico phenotype profiling. Drawing on the principle that similar gene expression patterns are likely to be associated with similar phenotype patterns, PhenoProfiler supplements the missing quantitative phenotype information for a given microarray dataset based on other well-characterized microarray datasets. We applied our method to 587 human microarray datasets covering >14,000 samples, and confirmed that the predicted phenotype profiles are highly consistent with true phenotype descriptions. PhenoProfiler offers several unique capabilities: (i) automated, multidimensional phenotype profiling, facilitating the analysis and treatment design of complex diseases; (ii) the extrapolation of phenotype profiles beyond provided classes; and (iii) the detection of confounding phenotype factors that could otherwise bias biological inferences. Finally, because no direct comparisons are made between gene expression values from different datasets, the method can use the entire body of cross-platform microarray data. This work has produced a compendium of phenotype profiles for the National Center for Biotechnology Information GEO datasets, which can facilitate an unbiased understanding of the transcriptome-phenome mapping. The continued accumulation of microarray data will further increase the power of PhenoProfiler, by increasing the variety and the quality of phenotypes to be profiled.

This paper introduces a health state modeling approach using clustering and Markov analysis to compare short- and long-term outcomes among health care populations. We provide a comparison to more conventional mixed effects regression methods and show that discrete state modeling offers a richer portrait of patient outcomes than the standard univariate techniques. We demonstrate our approach using primary data from a three year observational study of patients treated for schizophrenia at a VA Medical Center (VA) and in a Community Mental Health Center (CMHC) in the same urban community. Randomly selected samples of outpatients treated for schizophrenia or schizoaffective disorder were interviewed every six months using standardized psychiatric assessments such as the Positive and Negative Syndrome Scale (PANSS). Items from the PANSS were used to define 7 discrete health states representing different levels of severity and diverse mixtures of psychiatric symptoms. Conventional analysis showed that VA patients exhibited increasingly severe symptoms, while CMHC patients remained more stable over the study period. Health state analysis reinforced these results but also identified which subpopulations of VA patients were deteriorating. In particular they showed that there was little change over time among VA patients in the best and worst health states. Instead the deterioration was caused by VA patients with: a) mild symptoms and hallucinations and b) serious positive and negative symptoms, being more likely to enter a state with severe positive and negative symptoms accompanied by moderate general distress.

Motivation: In systems like Escherichia Coli, the abundance of sequence information, gene expression array studies and small scale experiments allows one to reconstruct the regulatory network and to quantify the effects of transcription factors on gene expression. However, this goal can only be achieved if all information sources are used in concert., Results: Our method integrates literature information, DNA sequences and expression arrays. A set of relevant transcription factors is defined on the basis of literature. Sequence data are used to identify potential target genes and the results are used to define a prior distribution on the topology of the regulatory network. A Bayesian hidden component model for the expression array data allows us to identify which of the potential binding sites are actually used by the regulatory proteins in the studied cell conditions, the strength of their control, and their activation profile in a series of experiments. We apply our methodology to 35 expression studies in E.Coli with convincing results., Availability: www.genetics.ucla.edu/labs/sabatti/software.html, Supplementary Information: The supplementary material are available at Bioinformatics online.

Objective: The objective of this study was to demonstrate a multivariate health state approach to analyzing complex disease data that allows projection of long-term outcomes using clustering, Markov modeling, and preference weights., Subjects: We studied patients hospitalized 30 to 364 days with refractory schizophrenia at 15 Veterans Affairs medical centers., Study Design: We conducted a randomized clinical trial comparing clozapine, an atypical antipsychotic, and haloperidol, a conventional antipsychotic., Methods: Health status instruments measuring disease-related symptoms and drug side effects were administered in face-to-face interviews at baseline, 6 weeks, and quarterly follow-up intervals for 1 year. Cost data were derived from Veterans Affairs records supplemented by interviews. K-means clustering was used to identify a small number of health states for each instrument. Markov modeling was used to estimate long-term outcomes., Results: Multivariate models with 7 and 6 states, respectively, were required to describe patterns of psychiatric symptoms and side effects (movement disorders). Clozapine increased the proportion of clients in states characterized by mild psychiatric symptoms and decreased the proportion with severe positive symptoms but showed no long-term benefit for negative symptoms. Clozapine dramatically increased the proportion of patients with no movement side effects and decreased incidences of mild akathisia. Effects on extrapyramidal symptoms and tardive dyskinesia were far less pronounced and slower to develop. Markov modeling confirms the consistency of these findings., Conclusions: Analyzing complex disease data using multivariate health state models allows a richer understanding of trial effects and projection of long-term outcomes. Although clozapine generates substantially fewer side effects than haloperidol, its impact on psychiatric aspects of schizophrenia is less robust and primarily involves positive symptoms.

Purpose: To determine if three-dimensional ultrasound (3D US), by nature of its ability to simultaneously evaluate structures in three orthogonal planes and to study relationships of devices to tumor(s) and surrounding anatomic structures from any desired orientation, adds significant additional information to real-time 2D US used for placement of devices for ablation of focal liver tumors., Materials and Methods: Sixteen patients underwent focal ablation of 23 liver tumors during two intraoperative cryoablation (CA) procedures, three intraoperative radiofrequency ablation (RFA) procedures, 11 percutaneous ethanol injections (PEI) procedures, and six percutaneous RFA procedures. After satisfactory placement of the ablative device(s) with 2D US guidance, 3D US was used to reevaluate adequacy to device position. Information added by 3D US and resultant alterations in device deployment were tabulated., Results: 3D US added information in 20 of 22 (91%) procedures and caused the operator to readjust the number or position of ablative devices in 10 of 22 (45%) of procedures. Specifically, 3D US improved visualization and confident localization of devices in 13 of 22 (59%) procedures, detected unacceptable device placement in 10 of 22 (45%), and determined that 2D US had incorrectly predicted device orientation to a tumor in three of 22 (14%)., Conclusions: Compared to conventional 2D US, 3D US provides additional relationship information for improved placement and optimal distribution of ablative agents for treatment of focal liver malignancy.

Purpose: To determine if three-dimensional (3D) ultrasonography (US) improves the ability to define the location and extent of facial clefting prenatally compared with two-dimensional (2D) US., Materials and Methods: Thirty-one fetuses suspected of having a facial cleft were examined prospectively with 2D and 3D US. Follow-up was performed in all fetuses., Results: Twenty-eight fetuses had a cleft lip at birth. The location of the cleft lip was correctly identified in all fetuses with 3D US and in 26 of 28 with 2D US. Twenty-two fetuses had a cleft primary palate. Nineteen and nine of 22 cleft palates were identified by using 3D and 2D US, respectively. Three fetuses suspected of having a facial cleft at 2D US had a normal palate at 3D US and at birth., Conclusion: Three-dimensional US is useful to identify the location and extent of facial clefting. The advantages of 3D US are the following: (a) The face may be viewed in a standard orientation, (b) the defect may be viewed systematically by using an interactive display, and (c) the rendered image provides landmarks for the planar images. Patient decisions may be affected, since they can view the abnormality on a recognizable 3D rendered image.

The aim of this investigation was to compare the utility of three-dimensional ultrasonography versus two-dimensional ultrasonography in imaging the neonatal brain. Thirty patients in the neonatal intensive care unit underwent two-dimensional and three-dimensional ultrasonography. The resultant two- and three-dimensional images recorded on film and three-dimensional volumes (reviewed on a workstation) were evaluated independently. Comparable numbers of normal and abnormal studies were diagnosed by each modality. Axial images were considered useful in approximately 50% of three-dimensional cases. Image quality, overall and in the far-field, was rated higher on two-dimensional images. Three-dimensional sonographic acquisition time in the neonatal intensive care unit (1.7 min+/-0.7 standard deviation) was significantly shorter than that for two-dimensional sonography (9.0+/-4.5 min). The total time for evaluation on the three-dimensional workstation (4.4+/-1.1 min) was significantly less than that for two-dimensional images on film (10.6+/-4.7 min). In conclusion, three-dimensional ultrasonography is a promising, diagnostically accurate, and efficient imaging tool for evaluation of the neonatal brain; however, visualization must improve before it can replace two-dimensional ultrasonography.

Purpose: To evaluate the usefulness of information provided by three-dimensional ultrasound (3D US) and to determine whether 3D US decreased the number of passes required to obtain portal vein (PV) access during creation of transjugular intrahepatic portosystemic shunts (TIPS)., Materials and Methods: Intermittent 3D US volume acquisitions were obtained during creation of TIPS in 20 patients. Useful information provided by 3D US was tabulated. The number of passes required to achieve PV access was recorded and results were compared retrospectively to 25 patients who underwent TIPS without 3D US., Results: 3D US documented that the operator's opinion of which hepatic vein had been selected was incorrect in nine patients (45%), detected unfavorable PV anatomy that required modification of equipment or technique in seven patients (35%), permitted estimation of the trajectory required to access the targeted PV in all patients (100%), assisted in selecting the optimal point along the hepatic vein for origination of the needle pass in 11 patients (55%), allowed avoidance of a large hepatocellular carcinoma in one patient (5%), and confirmed that access into the main PV was intrahepatic in four patients (20%). The mean number of needle passes decreased from 10.4 in the historic control group to 4.6 in the 3D US group (P = .0001)., Conclusion: 3D US provided imaging information that detected technical errors and altered anatomy, and provided positional and directional information to significantly improve needle pass efficiency.

Combined inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) is a candidate therapy for hypertension and cardiac failure. Given that NEP and ACE metabolize angiotensin (Ang) and bradykinin (BK) peptides, we investigated the effects of NEP inhibition and combined NEP and ACE inhibition on Ang and BK levels in rats with myocardial infarction. We administered the NEP inhibitor ecadotril (0, 0.1, 1, 10, and 100 mg/kg/day), either alone or together with the ACE inhibitor perindopril (0.2 mg/kg/day) by 12-hourly gavage from day 2 to 28 after infarction. Ecadotril increased urine cyclic GMP and BK-(1-9) excretion. Perindopril potentiated the effect of ecadotril on urine cyclic GMP excretion. Neither perindopril nor ecadotril reduced cardiac hypertrophy when administered separately, whereas the combination of perindopril and 10 or 100 mg/kg/day ecadotril reduced heart weight/body weight ratio by 10%. Administration of ecadotril to perindopril-treated rats decreased plasma Ang-(1-7) levels, increased cardiac BK-(1-9) levels, and increased Ang II levels in plasma, kidney, aorta, and lung. These data demonstrate interactions between the effects of NEP and ACE inhibition on remodeling of the infarcted heart and on Ang and BK peptide levels. Whereas increased cardiac BK-(1-9) levels may contribute to the reduction of cardiac hypertrophy, the reduction in plasma Ang-(1-7) levels and increase in Ang II levels in plasma and tissues may compromise the therapeutic effects of combined NEP/ACE inhibition.

The combination of neutral endopeptidase 24.11 (NEP) and angiotensin converting enzyme (ACE) inhibition is a candidate therapy for hypertension and cardiac failure. Given that NEP and ACE metabolize angiotensin (Ang) and bradykinin (BK) peptides, we investigated the effects of NEP inhibition and combined NEP and ACE inhibition on the levels of these peptides. We administered the NEP inhibitor ecadotril (0, 0.1, 1, 10, 100 mg/kg per day), either alone or together with the ACE inhibitor perindopril (0.2 mg/kg per day), to rats by 12 hourly gavage for 7 days. Ecadotril produced diuresis, natriuresis, increased urine cyclic guanosine monophosphate and BK-(1-9) levels, increased Ang II and Ang I levels in plasma, and increased Ang I levels in heart. Perindopril reduced Ang II levels in kidney, and increased BK-(1-9) levels in blood, kidney and aorta. Combined NEP/ACE inhibition produced the summation of these effects of separate NEP and ACE inhibition. In addition, perindopril potentiated the ecadotril-mediated diuresis, natriuresis and decrease in urine BK-(1-7)/BK-(1-9) ratio, which is an index of BK-(1-9) metabolism. Moreover, combined NEP/ACE inhibition increased Ang II levels in plasma and lung. These data indicate that summation of the effects of separate NEP and ACE inhibition provides the basis for the therapeutic efficacy of their combination. Whereas potentiation by perindopril of the diuretic and natriuretic effects of ecadotril may contribute to the therapeutic effects, increased Ang II levels in plasma and lung may compromise the therapeutic effects of combined NEP/ACE inhibition.

Dual inhibition of neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) offers the potential for improved therapy of hypertension and cardiac failure. S 21402-1 [(2S)-2-[(2S,3R)-2-thiomethyl-3-phenylbutanamido] propionic acid] is a sulfhydryl-containing potent inhibitor of both NEP (Ki = 1.7 nM) and ACE (Ki = 4.5 nM). S 21402-1 and the sulfhydryl-containing ACE inhibitor captopril were administered to rats by intraperitoneal injection (0, 0.3, 3, 30, 300 mg/kg). Urine was collected for 4 h; then plasma and kidneys were collected. The difference in NEP and ACE inhibition by S 21402-1 in vivo was greater than 1000-fold. All doses of S 21402-1 inhibited NEP, as indicated by plasma NEP activity, radioinhibitor binding to kidney sections, urinary sodium excretion and bradykinin-(1-7)/bradykinin-(1-9) ratio. However, only 300 mg/kg S 21402-1 inhibited ACE, as indicated by plasma angiotensin II/angiotensin I ratio, renin and angiotensinogen levels. Although S 21402-1 (30 and 300 mg/kg) inhibited renal NEP, as indicated by the bradykinin-(1-7)/bradykinin-(1-9) ratio in kidney, S 21402-1 had no effect on renal ACE, as indicated by the angiotensin II/angiotensin I ratio in kidney. Moreover, captopril was greater than 10-fold more potent than S 21402-1 as an ACE inhibitor in vivo. In separate experiments, the pressor response of anesthetized rats to angiotensin I showed more rapid decay in ACE inhibition by S 21402-1 than by captopril. These studies indicated that in vivo modification of S 21402-1 caused a much greater decrease in potency of ACE inhibition than NEP inhibition. Consequently, effective ACE inhibition by S 21402-1 required doses much higher than those required for NEP inhibition.

We investigated protein kinase C participation in the contractile response to 5-hydroxytryptamine (5-HT), and in the interaction between 5-HT and endothelin-1, in aortas from control and diabetic rats. Diabetic rats display attenuated reactivity to 5-HT (i.e., approximately 47% of control maximum). The protein kinase C inhibitor calphostin C (1 microM) significantly reduced responses to 5-HT only in aortas from control rats. In diabetic rats, maximum responses to 5-HT, in the presence of endothelin-1 (3 nM), were not significantly different to controls. The additional presence of calphostin C significantly reduced responses only in aortas from diabetic rats. These results may indicate an abnormality in the protein kinase C second messenger system during diabetes.

Vasoconstrictor responses to 5-hydroxytryptamine (5-HT), alpha-methyl-5-HT, endothelin-1, arachidonic acid and the thromboxane A2-mimetic U46619 ((15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid) were obtained in blood-perfused hindquarters of 6-week streptozotocin-diabetic rats. When compared to responses obtained in hindquarters of control rats, responses to 5-HT, alpha-methyl-5-HT, and arachidonic acid were attenuated in hindquarters of diabetic rats. However, responses to endothelin-1 or U46619 were not significantly different between controls and diabetics. These results suggest that 5-HT2, but not endothelin ETA receptor-mediated responses are reduced in hindquarters of diabetic rats. The results utilising arachidonic acid and U46619 suggest that there may also be a defect in the cyclo-oxygenase cascade during diabetes.

1. We have previously reported maximum responses to 5-hydroxytryptamine (5-HT) are diminished in endothelium-intact and -denuded aortae from rats with streptozotocin-induced diabetes of 2-weeks duration. 2. In the present study, the thromboxane A2/prostaglandin H2 (TP) receptor antagonist GR32191B (1 microM) significantly reduced maximum responses to 5-HT in endothelium-intact aortae from both control and diabetic rats. In the presence of GR32191B, maximum responses to 5-HT, in endothelium-intact aortae from diabetic rats, were still significantly reduced compared to those obtained in aortae from controls. 3. GR32191B (1 microM) had no significant effect on maximum responses to 5-HT in endothelium-denuded aortae from either control or diabetic rats. 4. Interaction between 5-HT (0.1 microM-0.1 mM) and threshold concentrations of endothelin-1 (ET-1) or the thromboxane (Tx)A2-mimetic, U46619, were examined in endothelium-intact and -denuded aortae from control and 2-week streptozotocin-diabetic rats. 5. Maximum responses to 5-HT in the presence of a threshold concentration of ET-1 (3 nM), in endothelium-intact aortae from diabetic rats, were not significantly different from those of control rats. 6. Maximum responses to 5-HT in the combined presence of ET-1 (3 nM) and GR32191B (1 microM), in endothelium-intact aortae from diabetic rats, were significantly reduced compared to those obtained in aortae from controls. 7. Maximum responses to 5-HT in the presence of ET-1 (3 nM) in endothelium-denuded aortae from diabetic rats were significantly reduced compared to those from controls. 8. Maximum responses to 5-HT in the presence of a threshold concentration of U46619 (20 or 30 nM),in endothelium-intact aortae from diabetic rats, were not significantly different from responses of controls.9. Maximum responses to 5-HT in the presence of a threshold (5-20 nM) concentration of U46619, in endothelium-denuded aortae from diabetic rats, were not significantly different from responses of controls.10 The results of the present study indicate that endothelial-derived TxA2 contributes to the contractile response to 5-HT in aortae from control and diabetic rats. Endothelial-derived TxA2 also appears to play a role in the potentiation of 5-HT responses by ET-1 in aortae from diabetic rats.

To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 mu and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100 degrees C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100 degrees C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.

1. This study was designed to examine further the attenuated contractile responses to 5-hydroxytryptamine (5-HT) previously observed in aortae from diabetic rats. 2. Cumulative concentration-response curves to 5-HT, and the 5-HT receptor agonists, alpha-methyl 5-HT (alpha-Me-5-HT, 5-HT2/1C agonist), (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2- aminopropane (DOI, 5-HT2/1C agonist) and 5-carboxamidotryptamine (5-CT, 5-HT1A/1B/1D agonist), were examined in endothelium-intact and -denuded aortae from 2-week streptozotocin (STZ)-diabetic and control rats. 3. In endothelium-intact and -denuded aortae from diabetic rats, maximum responses to 5-HT and alpha-Me-5-HT were significantly reduced compared to those of aortae from control rats. Responses to these agonists were inhibited by the 5-HT2/1C receptor antagonist, ketanserin (0.1 microM). 4. The attenuated responses to 5-HT of aortae from diabetic rats were normalized by chronic insulin treatment of the rats (5 units day-1, s.c.), but not by altering the glucose concentration of the bathing fluid. 5. The nitric oxide synthase inhibitor N-nitro-L-arginine (NOLA, 0.1 mM) significantly potentiated responses to both 5-HT and alpha-Me-5-HT in endothelium-intact aortae. However, the difference between maximum responses of aortae from diabetic and control rats was still evident in the presence of NOLA. 6. Endothelium-intact rings, in the presence of ketanserin (0.1 microM) and preconstricted with the thromboxane A2-mimetic, U46619 (0.1-0.3 microM), from control and diabetic rats, did not relax to cumulative additions of 5-HT (1 nM-30 microM). 7. Contractile responses to DOI were obtained only in endothelium-denuded aortae, and in endothelium-intact aortae in the presence of NOLA, from control rats.8. Contractile responses to 5-CT were obtained only in endothelium-denuded aortae from both control and diabetic rats, and in endothelium-intact aortae in the presence of NOLA, from control rats.9. [3H]-ketanserin binding studies showed that there was no significant change in the affinity or density of [3H]-ketanserin for binding sites in membrane preparations of aortae from control and diabetic rats.10. These results suggest that 5-HT contracts aortae from rats via 5-HT2/1c receptor activation.However, the simultaneous release of EDRF from endothelial cells in response to 5-HT does not appear to be receptor-mediated. The attenuated contractile responses observed to 5-HT in aortae from 2-week diabetic rats do not appear to be mediated by changes in either endothelial cell function or an alteration in 5-HT receptor affinity or density.

We investigated constrictor responses to 5-hydroxytryptamine (5-HT) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, a 5-HT2/5-HT1C receptor agonist) of aortic rings from 2- and 6-week streptozotocin-diabetic and vehicle control rats. At 10 g resting tension, maximum responses and -log EC50 values to 5-HT were significantly reduced in endothelium-intact and denuded aortas from 2- and 6-week diabetic rats relative to those from control rats (except for -log EC50 of endothelium-intact rings from 6-week diabetic rats). Removal of endothelium from aortas of 2- and 6-week diabetic and control rats caused significant increases both in -log EC50 values and in maximum responses to 5-HT. DOI caused marked contraction of endothelium-denuded aortas from control rats, but not of endothelium-intact aortas from control rats or aortas (either with or without endothelium) from diabetic rats. The nitric oxide (NO) synthase inhibitor N-nitro-L-arginine (NOLA) significantly potentiated constrictor responses to 5-HT in endothelium-intact aortas from control and diabetic rats. NOLA significantly potentiated constrictor responses to DOI in endothelium-intact aortas from control rats, but not in endothelium-intact aortas from diabetic rats. These results suggest that for aortas from 2- and 6-week diabetic rats, the diminished responses to 5-HT and DOI may be a result of reductions in 5-HT2-receptor-mediated responses of smooth muscle. The results also suggest that 5-HT and DOI can stimulate NO release from endothelial cells.