Accumulation of Somatic Mutations in Normal Human Colonic Epithelium

I investigated stem cell dynamics in normal human colon by detecting somatic variants affecting X-linked and autosomal genes using immunohistochemistry. Applying neutral clonal marks to a large cohort of patients to interpret age-related trends in clone frequencies has established the baseline stem cell dynamics of the tissue. Analysis of a number of new clonal marks showing biased behaviours suggests that different gene-specific mutations can subvert constraints resulting from the tissue architecture in different ways. In respect of intra-gland competition of stem cell derived clones, a disadvantage is observed for the histone modifier HDAC6, while at the other end of the spectrum, loss of the cohesin member STAG2 strongly advantages affected stem cells. Subsequent clonal expansion beyond the boundary of a single crypt is recognised by clones occupying multi-crypt patches. Quantification of such events allows the rate of lateral expansion for different mutations to be measured. Moderate effects were found for PTEN, p53 and STAG2, while mutations in the histone demethylase KDM6A generate very large areas of mutant epithelium in aged humans. Further, targeted sequencing revealed dramatic expansion of KRAS-mutant clones in histologically normal colon. Patches may arise from crypt fission and fusion events. Using a clonal mark based on mild periodic acid-Schiff staining, the neutral crypt fission and fusion rates were quantified. Against this baseline, it was found that KDM6A-mutant clones expand mainly by fission, while fusion remains at homeostatic levels. The emerging picture is that of the aged human colon consisting of a mosaic of different mutations. The work presented in this thesis offers detailed insights into the rates at which different gene-specific mutations arising in colonic stem cells can become fixed within individual crypts and undergo subsequent lateral expansion through crypt fission and fusion events. This defines the timeframe taken for cancer drivers to achieve high mutant allele burden within the tissue, which can serve as a basis for cancer prevention strategies.Wellcome Trus

PREVALENCE OF 'BORDERLINE' VALUES OF CARDIOVASCULAR RISK FACTORS IN THE CLINICAL PRACTICE OF GENERAL MEDICINE IN ITALY: RESULTS OF THE BORDERLINE STUDY.

INTRODUCTION: The prevalence of patients with 'borderline' levels of cardiovascular risk factors has been rarely investigated, being often reported in studies evaluating abnormal values of these parameters. The BORDERLINE study represents a pilot experience to primarily identify the prevalence of 'high-normal' conditions, such as pre-hypertension, lipid and glucose levels in the upper range of normality in the setting of general practice in Italy. AIM: The aim of this study was to evaluate the prevalence of patients with 'borderline' values of cardiovascular risk factors in Italy. METHODS: Involved physicians were asked to evaluate the first 20 outpatients, consecutively seen in June 2009. Data were collected in a study-designed case-report form, in which physicians identified thresholds rather than reported absolute values of several clinical parameters. High-normal values were defined as follows: blood pressure (BP) 130-140/85-90 mmHg; total cholesterol 180-200 mg/dL; low-density lipoprotein cholesterol (LDL-C) 130-150 mg/dL; high-density lipoprotein cholesterol (HDL-C) 30-40 mg/dL in males and 40-50 mg/dL in females; triglycerides 130-150 mg/dL and fasting glucose 100-110 mg/dL. RESULTS: Fifty-three Italian physicians provided valuable clinical data on 826 individual outpatients, among which 692 (83.7%, 377 women, mean age 60.9 ± 13.2 years, body mass index 26.6 ± 5.0 kg/m2) were included in the present analysis. Prevalence of borderline values of systolic BP and total cholesterol levels were at least comparable with those in the normal limits of the corresponding parameters, whereas prevalence of borderline diastolic BP, LDL-C, HDL-C, triglycerides and fasting glucose levels was significantly lower than that of normal values, but higher than that of abnormal values of the corresponding parameters. CONCLUSIONS: Using this sample of healthy subjects in the setting of general practice in Italy, our results demonstrated a relatively high prevalence of borderline values of cardiovascular risk factors, which was at least comparable with that of normal, but significantly higher than that of abnormal thresholds. These preliminary findings may prompt more extensive investigations in the area of 'borderline' cardiovascular risk. This information may, in fact, potentially enable the design of more effective prevention strategies in the future to limit the burden of cardiovascular disease in the general population in Italy

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred

Changes in cortical [beta]-adrenergic receptor density and neuronal sensitivity to norepinephrine accompany morphine dependence and withdrawal

Radioligand binding experiments were carried out in conjunction with electrophysiological recordings in vivo in the parietal cortex in rats to assess changes in postsynaptic [beta]-adrenergic receptor function that result after chronic administration of morphine and during morphine withdrawal. Chronic treatment of rats with morphine for 14 days resulted in a 38% increase in the density of [beta]-adrenergic receptors in the parietal cortex, as measured by the binding of the specific antagonist [3H]dihydroalprenolol (DHA). In comparison, following withdrawal in the chronic morphine-treated animals, the number of specific [3H]DHA binding sites in this same cortical region was decreased 25%, when compared to saline-treated controls. These alterations in cortical [beta]-adrenergic receptor density were not accompanied by a significant change in the dissociation constant (Kd) for [3H]DHA or in the inhibitory constants (Ki) for the specific agonists norepinephrine and isoproterenol. Microiontophoretic testing revealed that the changes in [beta]-adrenergic receptor density found in parietal cortex after chronin morphine treatment and during morphine withdrawal were accompanied by a selective increase and decrease, respectively, in the sensitivity of cerebrocortical neurons in the same region to [beta]-adrenergic stimulation. These results suggest that changes in central adrenergic function might be related to the formation and/or expression of dependence on morphine.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26883/1/0000449.pd

Gastrin-Releasing Peptide Signaling Plays a Limited and Subtle Role in Amygdala Physiology and Aversive Memory

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe6, Leu-NHEt13, des-Met14)-Bombesin (6–14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders

Disrupted Functional Connectivity with Dopaminergic Midbrain in Cocaine Abusers

Background: Chronic cocaine use is associated with disrupted dopaminergic neurotransmission but how this disruption affects overall brain function (other than reward/motivation) is yet to be fully investigated. Here we test the hypothesis that cocaine addicted subjects will have disrupted functional connectivity between the midbrain (where dopamine neurons are located) and cortical and subcortical brain regions during the performance of a sustained attention task. Methodology/Principal Findings: We measured brain activation and functional connectivity with fMRI in 20 cocaine abusers and 20 matched controls. When compared to controls, cocaine abusers had lower positive functional connectivity of midbrain with thalamus, cerebellum, and rostral cingulate, and this was associated with decreased activation in thalamus and cerebellum and enhanced deactivation in rostral cingulate. Conclusions/Significance: These findings suggest that decreased functional connectivity of the midbrain interferes with the activation and deactivation signals associated with sustained attention in cocaine addicts

Postnatal ontogeny of GABAB binding in rat brain

The postnatal development of GABAB binding sites in rat brain was studied by quantitative receptor autoradiography using [3H]GABA under selective conditions. Binding levels peak at regionally specific times during the first three weeks of life and then decline to adult levels. GABAB binding peaked in the globus pallidus, vestibular and spinal trigeminal nuclei, and the CA3 region of the hippocampus at postnatal day 3; in the striatum, nucleus accumbens, inferior olive, septum, dentate gyrus and CA1 region of the hippocampus at postnatal day 7; in the neocortex and thalamus at postnatal day 14; and in the medial geniculate at postnatal day 21. Following these regionally specific peaks, binding decreased to postnatal day 28 levels. Further significant decreases in binding were observed in all regions examined between postnatal day 28 and adulthood. Comparisons of binding site pharmacology reveal equipotent displacement of GABAB binding by several competitive agonists and antagonists in postnatal day 7 and adult rat brain, indicating that immature and adult binding sites have similar pharmacological properties with regard to these compounds. The GABAB receptor antagonist CGP 54626A, however, inhibited binding more potently in the postnatal day 7 thalamus and neocortex than in these areas in the adult brain. The guanyl nucleotide analogue guanosine 5'-O-(3-thiotriphasphate) inhibited GABAB binding extensively in both postnatal day 7 and adult brain. The non-competitive antagonist zinc also inhibited GABAB binding at both ages and was more potent in postnatal day 7 brain than in adult brain. Saturation analyses reveal two binding sites with similar affinities in both immature and adult rat brain, indicating that postnatal modulation of GABAB binding reflects changes in binding site density rather than modulation of binding site affinity. While immature GABAB binding sites share most pharmacological characteristics with adult binding sites and appear to be coupled to G-proteins at an early age, their interactions with zinc and CGP 54626A suggest that GABAB binding sites in immature brain may have a distinct pharmacological profile.Our data suggest significant regional and pharmacological changes in GABAB binding during development. The implications of these findings are discussed with regards to a possible role of GABAB receptors in the development of the central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31372/1/0000285.pd

[ 18 F]fluoroethoxy-benzovesamicol, a PET radiotracer for the vesicular acetylcholine transporter and cholinergic synapses

Loss of cholinergic transmission in the cortex and hippocampus is a characteristic feature of Alzheimer's disease, and visualization of functional cholinergic synapses in the brain with PET could be a useful method for studying this degenerative condition in living humans. We investigated [ 18 F]fluoroethoxybenzovesamicol, (−)-[ 18 F]FEOBV, (−)-(2R,3R)-trans-2-hydroxy-3-(4-phenylpiperidino)-5-(2-[ 18 F]fluoroethoxy)-1,2,3,4-tetralin, a high affinity positron emitting ligand for the vesicular acetylcholine transporter, as a potential in vivo cholinergic synapse mapping agent. Rodent biodistribution, dosimetry, stereospecificity of biological effects, pharmacologic blocking studies, in vivo rodent brain autoradiography and metabolites were examined. (−)-[ 18 F]FEOBV brain uptake following intravenous injection was robust, with 2.65% dose/brain in mice at 5 min, and the regional localization matched the known distributions of presynaptic cholinergic markers at later times. Both the cholinergic localization and curare-like effects of FEOBV were associated with the “(−)”-enantiomer exclusively. (−)-[ 18 F]FEOBV regional brain distribution in rodents was changed little by pretreatment with haloperidol, (+)-3-PPP, or E-2020, indicating FEOBV, unlike other vesamicol analogs, did not interact in vivo with dopamine or σ receptor systems. Autoradiography of rat brain 3 h following i.v. injection of (−)-[ 18 F]FEOBV showed high localization in brain areas rich in presynaptic cholinergic elements. Metabolic defluorination in rodents was modest, and analysis of brain tissue following tracer administration found FEOBV as the only extractable radioactive species. (−)-[ 18 F]FEOBV dosimetry calculated from rat data estimate 10 mCi doses can be given to humans. These studies show FEOBV maps cholinergic areas with high specificity in vivo, and may provide a noninvasive means to safely and accurately gauge the functional integrity of cholinergic synapses in man using PET. Synapse 30:263–274, 1998. © 1998 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34984/1/4_ftp.pd