Paul Bentley

Effects of Cholinergic Enhancement on Visual Stimulation, Spatial Attention, and Spatial Working Memory

We compared behavioral and neural effects of cholinergic enhancement between spatial attention, spatial working memory (WM), and visual control tasks, using fMRI and the anticholinesterase physostigmine. Physostigmine speeded responses nonselectively but increased accuracy selectively for attention. Physostigmine also decreased activations to visual stimulation across all tasks within primary visual cortex, increased extrastriate occipital cortex activation selectively during maintained attention and WM encoding, and decreased parietal activation selectively during maintained attention. Finally, lateralization of occipital activation as a function of the visual hemifield toward which attention or memory was directed was decreased under physostigmine. In the case of attention, this effect correlated strongly with a decrease in a behavioral measure of selective spatial processing. Our results suggest that, while cholinergic enhancement facilitates visual attention by increasing activity in extrastriate cortex generally, it accomplishes this in a manner that reduces expectation-driven selective biasing of extrastriate cortex.

Cholinergic enhancement modulates neural correlates of selective attention and emotional processing

Neocortical cholinergic afferents are proposed to influence both selective attention and emotional processing. In a study of healthy adults we used event-related fMRI while orthogonally manipulating attention and emotionality to examine regions showing effects of cholinergic modulation by the anticholinesterase physostigmine. Either face or house pictures appeared at task-relevant locations, with the alternative picture type at irrelevant locations. Faces had either neutral or fearful expressions. Physostigmine increased relative activity within the anterior fusiform gyrus for faces at attended, versus unattended, locations, but decreased relative activity within the posterolateral occipital cortex for houses in attended, versus unattended, locations. A similar pattern of regional differences in the effect of physostigmine on cue-evoked responses was also present in the absence of stimuli. Cholinergic enhancement augmented the relative neuronal response within the middle fusiform gyrus to fearful faces, whether at attended or unattended locations. By contrast, physostigmine influenced responses in the orbitofrontal, intraparietal and cingulate cortices to fearful faces when faces occupied task-irrelevant locations. These findings suggest that acetylcholine may modulate both selective attention and emotional processes through independent, region-specific effects within the extrastriate cortex. Furthermore, cholinergic inputs to the frontoparietal cortex may influence the allocation of attention to emotional information.

Cholinergic modulation of cognition: insights from human pharmacological functional neuroimaging.

Highlights • We review all 63 healthy human cholinergic functional neuroimaging studies to date. • General findings from fMRI and PET studies are synthesised with biological models. • Cholinergic neuromodulations are divided into sensory, attentional or memory. • Methodological issues of pharmacological functional imaging are discussed.

Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia.

Although advances have been made in the development of more effective treatment modalities, B‐cell chronic lymphocytic leukaemia (B‐CLL) remains incurable due to the development of drug resistance. Defective programmed cell death mechanisms rather than dysregulation of cell cycle appears to predominate in B‐CLL and it is likely that a failure to initiate apoptosis contributes to chemoresistance. Most B‐CLL cells contain high levels of the anti‐apoptotic protein Bcl‐2 and high Bcl‐2/Bax ratios have been associated with in vitro resistance to cytotoxic agents. In this study we evaluated the cellular responses to a Bcl‐2 antisense oligonucleotide in terms of Bcl‐2 mRNA and protein expression and the induction of apoptosis. The antisense molecule induced a specific reduction in Bcl‐2 mRNA and protein expression over the 48 h culture period and was associated with increased apoptosis. The study indicates that Bcl‐2 protein is central to the mediation of resistance to apoptosis in B‐CLL. Therefore Bcl‐2 antisense oligonucleotides might be useful in the treatment of B‐CLL.

Common polymorphism G(-248)A in the promoter region of the bax gene results in significantly shorter survival in patients with chronic lymphocytic Leukemia once treatment is initiated.

PURPOSE Chronic lymphocytic leukemia (CLL) is characterized by the development of drug resistance. The underlying biologic and genetic reasons for this resistance are complex, but the bcl-2 gene family seems to play a critical role. This retrospective study assessed the clinical impact of a common single nucleotide polymorphism of the pro-apoptotic bax gene in patients with chronic lymphocytic leukemia. PATIENTS AND METHODS The frequency of the novel polymorphism, G(-248)A, in the promoter region of the bax gene and bax protein expression was assessed in 203 CLL patients. The results were correlated with clinical outcome. RESULTS The polymorphism was found in 23% of the CLL cohort and 15% of normal controls with no significant difference in allele frequency between the two groups (P = .15). It was associated with lower Bax protein expression and a shorter overall survival, especially in the treated patient group (P = .03). Furthermore, the adverse impact of the polymorphism was accentuated when comparing survival from the date of first treatment rather than diagnosis (P = .012). No significant difference in age at diagnosis, stage of disease at presentation, lymphocyte doubling time, time to first treatment, or progression-free survival were observed. CONCLUSION The presence of this single nucleotide polymorphism in CLL critically influences the response to treatment and overall survival. Given the relatively high prevalence of this polymorphism in the normal population, further prospective studies in CLL and other human malignancies are indicated.

Causal Relationship of Susceptibility Genes to Ischemic Stroke: Comparison to Ischemic Heart Disease and Biochemical Determinants

Interrelationships between genetic and biochemical factors underlying ischemic stroke and ischemic heart disease are poorly understood. We: 1) undertook the most comprehensive meta-analysis of genetic polymorphisms in ischemic stroke to date; 2) compared genetic determinants of ischemic stroke with those of ischemic heart disease, and 3) compared effect sizes of gene-stroke associations with those predicted from independent biochemical data using a mendelian randomization strategy. Electronic databases were searched up to January 2009. We identified: 1) 187 ischemic stroke studies (37,481 cases; 95,322 controls) interrogating 43 polymorphisms in 29 genes; 2) 13 meta-analyses testing equivalent polymorphisms in ischemic heart disease; and 3) for the top five gene-stroke associations, 146 studies (65,703 subjects) describing equivalent gene-biochemical relationships, and 28 studies (46,928 subjects) describing biochemical-stroke relationships. Meta-analyses demonstrated positive associations with ischemic stroke for factor V Leiden Gln506, ACE I/D, MTHFR C677T, prothrombin G20210A, PAI-1 5G allele and glycoprotein IIIa Leu33Pro polymorphisms (ORs: 1.11 - 1.60). Most genetic associations show congruent levels of risk comparing ischemic stroke with ischemic heart disease, but three genes--glycoprotein IIIa, PAI-1 and angiotensinogen--show significant dissociations. The magnitudes of stroke risk observed for factor V Leiden, ACE, MTHFR and prothrombin, but not PAI-1, polymorphisms, are consistent with risks associated with equivalent changes in activated protein C resistance, ACE activity, homocysteine, prothrombin, and PAI-1 levels, respectively. Our results demonstrate causal relationships for four of the most robust genes associated with stroke while also showing that PAI-1 4G/5G polymorphism influences cardiovascular risk via a mechanism not simply related to plasma levels of PAI-1 (or tPA) alone.

Flow cytometric assessment of three different methods for the measurement of in vitro apoptosis

Chlorambucil-induced apoptosis was assessed by three different flow cytometric methods in B-cell chronic lymphocytic leukaemia (B-CLL) cells cultured in vitro and the results were compared with those derived from the morphological assessment of the same samples. Spontaneous apoptosis was consistently observed in the control cultures in the absence of drug but this accounted for less than 12% of all cells in every case. The methods under investigation were the Annexin V labelling assay, the terminal deoxynucleotidyl transferase (TdT) end-labelling assay and the labelling of a 38 kDa mitochondrial membrane protein (7A6 antigen) which is exposed on cells undergoing apoptotic cell death (Apo2.7 assay). The Annexin V assay consistently stained a higher percentage of cells and with a greater separation between the positive and negative cell populations. We conclude that the phosphatidyl serine translocation to the outer leaflet of the cell membrane following an apoptotic signal, as labelled by Annexin V, probably occurs before the development of the DNA strand breaks or the exposure of 7A6 antigen in those cells triggered to die by apoptosis.

Flavopiridol circumvents Bcl‐2 family mediated inhibition of apoptosis and drug resistance in B‐cell chronic lymphocytic leukaemia

Flavopiridol, a synthetic flavone, is currently under clinical investigation for the treatment of B‐cell chronic lymphocytic leukaemia (B‐CLL). In this study, we examined the in vitro effects of flavopiridol and fludarabine on B‐CLL cells from 64 patients (36 treated and 28 untreated) in terms of apoptosis induction and Bcl‐2 family expression. Both flavopiridol and fludarabine induced apoptosis in all the samples tested with mean LD50 values (± SD) of 59·7 nmol/l (± 36·5) and 6·2 μmol/l (± 7·5) respectively. Mean flavopiridol LD50 values were not significantly different between the treated and untreated patient groups (P = 0·35), whereas the fludarabine LD50 values were significantly higher in the previously treated patient group (P = 0·01). Bcl‐2 and Mcl‐1 expression were downregulated in both flavopiridol and fludarabine‐induced apoptotic cells, but the increase in Bax expression that accompanied fludarabine‐induced apoptosis was not evident in flavopiridol‐treated cells. In addition, Bcl‐2:Bax ratios were not predictive of flavopiridol cytotoxicity (P = 0·82), whereas they were highly predictive of in vitro responsiveness to fludarabine (P = 0·001). Overall, these findings suggest that flavopiridol exerts its cytotoxic effect through a novel cell‐death pathway that is not subject to the Bcl‐2 family mediated resistance mechanisms that reduce the efficacy of many conventional chemotherapeutic drugs.

Towards the identification of blood biomarkers for acute stroke in humans: a comprehensive systematic review

AIMS Identification of biomarkers for stroke will aid our understanding of its aetiology, provide diagnostic and prognostic indicators for patient selection and stratification, and play a significant role in developing personalized medicine. We undertook the largest systematic review conducted to date in an attempt to characterize diagnostic and prognostic biomarkers in acute ischaemic and haemorrhagic stroke and those likely to predict complications following thrombolysis. METHODS A comprehensive literature search was carried out to identify diagnostic and prognostic stroke blood biomarkers. Mean differences (MDs) and 95% confidence intervals (CIs) were calculated for each biomarker. RESULTS We identified a total of 141 relevant studies, interrogating 136 different biomarkers. Three biomarkers (C-reactive protein, P-selectin and homocysteine) significantly differentiated between ischaemic stroke and healthy control subjects. Furthermore, glial fibrillary acidic protein levels were significantly different between haemorrhagic stroke and ischaemic stroke patients (MD 224.58 ng l⁻¹; 95% CI 25.84, 423.32; P= 0.03), high levels of admission glucose were a strong predictor of poor prognosis after ischaemic stroke and symptomatic intracerebral haemorrhage post-thrombolysis, glutamate was found to be an indicator of progressive (unstable) stroke (MD 172.65 µmol l⁻¹, 95% CI 130.54, 214.75; P= 0.00001), D-dimer predicted in-hospital death (MD 0.67 µg ml⁻¹, 95% CI 0.35, 1.00; P= 0.0001), and high fibrinogen levels were associated with poor outcome at 3 months (MD 47.90 mg l⁻¹, 95% CI 14.88, 80.93; P= 0.004) following ischaemic stroke. CONCLUSIONS Few biomarkers currently investigated have meaningful clinical value. Admission glucose may be a strong marker of poor prognosis following acute thrombolytic treatment. However, molecules released in the bloodstream before, during or after stroke may have potential to be translated into sensitive blood-based tests.

Intra-Arterial Immunoselected CD34+ Stem Cells for Acute Ischemic Stroke

Treatment with CD34+ hematopoietic stem/progenitor cells has been shown to improve functional recovery in nonhuman models of ischemic stroke via promotion of angiogenesis and neurogenesis. We aimed to determine the safety and feasibility of treatment with CD34+ cells delivered intra‐arterially in patients with acute ischemic stroke. This was the first study in human subjects. We performed a prospective, nonrandomized, open‐label, phase I study of autologous, immunoselected CD34+ stem/progenitor cell therapy in patients presenting within 7 days of onset with severe anterior circulation ischemic stroke (National Institutes of Health Stroke Scale [NIHSS] score ≥8). CD34+ cells were collected from the bone marrow of the subjects before being delivered by catheter angiography into the ipsilesional middle cerebral artery. Eighty‐two patients with severe anterior circulation ischemic stroke were screened, of whom five proceeded to treatment. The common reasons for exclusion were age >80 years (n = 19); medical instability (n = 17), and significant carotid stenosis (n = 13). The procedure was well tolerated in all patients, and no significant treatment‐related adverse effects occurred. All patients showed improvements in clinical functional scores (Modified Rankin Score and NIHSS score) and reductions in lesion volume during a 6‐month follow‐up period. Autologous CD34+ selected stem/progenitor cell therapy delivered intra‐arterially into the infarct territory can be achieved safely in patients with acute ischemic stroke. Future studies that address eligibility criteria, dosage, delivery site, and timing and that use surrogate imaging markers of outcome are desirable before larger scale clinical trials.