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Aim: To investigate whether there is an association between subgingival microbial diversity and reduced respiratory function., Materials and Methods: A group of dentate 58-72-year-old men in Northern Ireland had a comprehensive periodontal examination including subgingival plaque sampling. DNA was extracted from plaque samples and the V1-V3 regions of the 16S rRNA gene were analysed by high-throughput sequencing and a microbial diversity index (MDI) was derived. Spirometry measurements were made using a wedge bellows spirometer. The primary outcome variable of interest was the percentage of predicted forced expiratory volume in 1 s (% predicted FEV 1 ). Analysis included multiple linear regression with adjustment for various confounders., Results: Five-hundred and seven men were included in the analysis. The mean age was 63.6 years (SD = 3.1). Of these, 304 (60.0%) men had no or mild periodontitis, 105 (20.7%) had moderate periodontitis and 98 (19.3%) had severe periodontitis. Multiple linear regression analysis showed that a one unit increase in MDI was associated with a 0.71% loss (95% confidence interval: 0.06%-1.35%; p = .03) in % predicted FEV 1 after adjustment for all confounders., Conclusions: In this group of dentate men from Northern Ireland, subgingival microbial diversity was associated with reduced respiratory function., (© 2023 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.)

Objective: To investigate the association between denture wearing and airflow limitation in men in Northern Ireland enrolled in the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study., Methods: A case-control design was used to study partially dentate men. Cases were men aged 58-72 years who were confirmed as denture wearers. Controls were never denture wearers who were matched by age (± 1 month) and smoking habit to the cases. The men had a periodontal assessment and completed a questionnaire detailing their medical history, dental history and behaviours, social circumstances, demographic background and tobacco use. Physical examination and spirometry measurements of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) were also undertaken. Spirometry data for edentulous men who wore complete dentures were compared with that recorded for the partially dentate men studied., Results: There were 353 cases who were partially dentate and were confirmed denture wearers. They were matched for age and smoking habit to never denture wearer controls. The cases had an FEV1 that was on average 140 ml lower than the controls, p = 0.0013 and a 4% reduction in percent predicted FEV1, p = 0.0022. Application of the GOLD criteria indicated that 61 (17.3%) of the cases had moderate to severe airflow limitation compared with 33 (9.3%) of controls, p = 0.0051. Fully adjusted multivariable analysis showed that partially dentate men who were denture wearers were significantly more likely (p = 0.01) to have moderate to severe airflow reduction with an adjusted odds ratio (OR) of 2.37 (95% confidence intervals 1.23-4.55). In the 153 edentulous men studied moderate to severe airflow limitation was recorded in 44 (28.4%), which was significantly higher than in the partially dentate denture wearers (p = 0.017), and the men who had never worn a denture (p<0.0001)., Conclusion: Denture wearing was associated with an increased risk of moderate to severe airflow limitation in the cohort of middle-aged Western European men studied., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kelly et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Objectives: Focused thoracic ultrasound (TUS) provides an increased safety profile when undertaking invasive pleural procedures. This has led to the requirement for defined curricula, high quality teaching and robust, validated assessment tools among physicians to ensure patient safety and clinical excellence. Current UK practice is based almost exclusively on expert consensus, but assessment methods employed have been shown to have low reliability and validity and are potentially open to bias. As a result, several assessment tools have been developed, although each has its own limitations., Methods: This study aimed to develop and validate an assessment tool corresponding to those skills associated with the most basic level of practice, defined recently as an emergency level operator in the British Thoracic Society Training Standards for Thoracic Ultrasound., Results: A total of 27 candidates were enrolled by two examiners based in Belfast and Oxford over a 10-month period between February and November 2019. Mean score of the inexperienced group was 44.3 (95% CI 39.2-49.4, range 28-54) compared with 74.9 (95% CI 72.8-77, range 64-80) in the experienced group providing an estimated mean difference of 30.7 between the two groups (95% CI 24.7-36.7; p < .001)., Conclusions: This tool appears to discriminate between trainees with limited experience of TUS performance and those with no experience. It has the potential to form part of the assessment strategy for trainees in the United Kingdom and beyond, alongside well established assessment tools in postgraduate training., (© 2022 Wiley Periodicals LLC.)

Background: In this "Advancing simulation practice" article, we offer an expose of the involvement of real patients in Objective Structured Clinical Examinations (OSCEs), inviting educators who traditionally involve solely SPs in their summative OSCEs to consider the practice. The need for standardisation in summative assessments can make educators understandably wary to try this, even if the rhetoric to involve real patients is accepted. We offer this as an instance of the tussle between standardisation and validity experienced throughout health professions education., Main Text: We offer our experience and empirical evidence of this simulation practice, based on an institutional ethnographic examination of the involvement of real patients in summative OSCEs from an undergraduate medical school in the UK. Our critique demonstrates the merits of this approach as an assessment environment closer to the real clinical environments where these soon-to-be doctors interact in a more authentic way with real patients and their illness experiences. We balance this against the extra work required for all involved and suggest the biggest challenge is in the reorientation work required for both Faculty and students who are institutionalised to expect standardisation above all in assessment., Conclusion: We advocate for involving real patients in summative OSCEs and hope that readers may feel compelled and empowered to foster this shift in mindset required to introduce this practice into their assessments., (© 2022. The Author(s).)

Aim: The aim was to investigate the role of systemic inflammation in the relationship between periodontitis, edentulism, and all-cause mortality in a group of men in Northern Ireland aged 58-72 years., Materials and Methods: A representative sample of 1558 men had a detailed dental examination between 2001 and 2003. The primary end point was death from any cause. Cox's proportional hazards model was used to assess the longitudinal relationship between periodontitis, edentulism, and all-cause mortality. Accelerated failure time modelling was performed to investigate the mediating role of systemic inflammation., Results: Mean age of the men at baseline was 64.3 (standard deviation 2.9) years. During a median follow-up of 17 years, 500 (32.1%) men died. After adjustment for confounding variables, compared to men with no/mild periodontitis, edentulous men had a hazard ratio for all-cause mortality of 1.52 (95% confidence interval [CI] 1.16-1.99) p < .01 and for those with severe periodontitis, it was 1.34 (95% CI 1.06-1.70) p = .01. Systemic inflammation accounted only for a minor mediating pathway effect of 10%., Conclusions: There was evidence in this group of men that those who were edentulous or had severe periodontitis had a significantly increased risk of all-cause mortality. Systemic inflammation was not a major explanatory mediator of this association., (© 2021 The Authors. Journal of Clinical Periodontology published by John Wiley & Sons Ltd.)

Introduction: Objective structured clinical examinations (OSCEs) are a complex form of assessment, where candidates can interact with 'patients' in a constructed socio-clinical encounter. Conceptualizing OSCEs as a complex socially and culturally situated activity offers important research affordances. There are concerns that OSCEs may encourage more strategic 'tick-box' candidate behaviours and have a potential negative impact on learner identity formation. This study explored, at a micro-level, the social roles and behaviours occurring within the OSCE triad of simulated patients, candidates and examiners. We used a theoretical framework drawn from Goffman's dramaturgy metaphor., Methods: OSCE candidates, examiners and simulated patients were invited, consented and recruited using maximal variation sampling. Participants were allocated to a summative OSCE circuit that had unobtrusive video cameras. Video footage of 18 stations was transcribed. Analysis was interpretative and iterative until a rich and thick description was achieved., Results: Focusing on elements of Goffman's dramaturgy metaphor, we foregrounded our analysis by considering the performers, costumes, props and the theatre of the OSCE. A combination of symbols, both physical and semiotic, was used to construct and maintain layered roles and identities within this tightly defined socio-clinical setting. Informed by this foregrounding, we then considered the social interactions and behaviours within the OSCE: 'Creating the right impression?', 'A performance of contradictions?' and 'Simulated patients: patients or props?', Discussion: In the pursuit of standardization, OSCEs have potential to mediate less desirable test-taking behaviours that are not entirely patient-centric, and beyond this may have an impact on professional identity. Whilst OSCE checklists provide objectivity, they have potential to promote a presentation of self that is in tension with good medical practice. The certainty of checklists needs to be looked at afresh in order to better reflect the many uncertainties that doctors face in real clinical practice. This research opens up new ways of thinking and enhancing future assessment practices.

Focused thoracic ultrasound has become essential in the guidance and direction of pleural interventions to reduce unwanted complications and as a result now forms a crucial component of physician training. Current training standards along with assessment methods vary widely, and are often not robust enough to ensure adequate competence.This review assesses the current state of training and assessment of thoracic ultrasound competence in various settings, allowing comparison with alternative competency based programmes. Future directions for training and assessment of thoracic ultrasound competence are discussed., Competing Interests: Conflict of interest: D.J. McCracken has nothing to disclose. Conflict of interest: C.B. Laursen has nothing to disclose. Conflict of interest: G. Barker has nothing to disclose. Conflict of interest: F.V. Gleeson has nothing to disclose. Conflict of interest: K.M. Cullen has nothing to disclose. Conflict of interest: N.M. Rahman has nothing to disclose., (Copyright ©ERS 2019.)

Cerebrovascular pathology is common in aging and Alzheimer's disease (AD). The microvasculature is particularly vulnerable, with capillary-level microhemorrhages coinciding with amyloid beta deposits in senile plaques. In the current analysis, we assessed the relationship between cerebral microvessels and the neuritic component of the plaque in cortical and hippocampal 50- to 200-μm sections from 11 AD, 3 Down syndrome, and 7 nondemented cases in neuritic disease stages 0-VI. We report that 77%-97% of neuritic plaques are perivascular, independently of disease stage or dementia diagnosis. Within neuritic plaques, dystrophic hyperphosphorylated tau-positive neurites appear as clusters of punctate, bulbous, and thread-like structures focused around capillaries and colocalize with iron deposits characteristic of microhemorrhage. Microvessels within the neuritic plaque are narrowed by 1.0 ± 1.0 μm-4.4 ± 2.0 μm, a difference of 16%-65% compared to blood vessel segments with diameters 7.9 ± 2.0-6.4 ± 0.8 μm (p < 0.01) outside the plaque domain. The reduced capacity of microvessels within plaques, frequently below patency, likely compromises normal microlocal cerebrovascular perfusion. These data link the neuritic and amyloid beta components of the plaque directly to microvascular degeneration. Strategies focused on cerebrovascular antecedents to neuritic dystrophy in AD have immediate potential for prevention, detection, and therapeutic intervention., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate whether there was an association between chronic periodontitis (CP) and reduced respiratory function., Methods: A group of dentate 58- to 72-year-old men in Northern Ireland had a comprehensive periodontal examination. Parallel to the periodontal examination, participants completed questionnaires gathering information on their medical history, social circumstances, demographic background and tobacco use. A physical examination assessed anthropometric measures. Fasting blood samples were obtained and analysed for high-sensitivity C-reactive protein (hs-CRP). Spirometry measures were performed using a wedge bellows spirometer (Vitalograph S Model). The primary outcome variable of interest was the percentage predicted forced expiratory volume in one-second (% predicted FEV 1 ). Analysis included multiple linear regression with adjustment for various confounders and a regression-based mediation analysis., Results: A total of 1,380 men were included in the analysis. The mean age was 63.7 years (SD 3.0). Multiple linear regression analysis showed that a doubling in mean clinical attachment loss (CAL) equated to a -3.33% (95% CI: -4.80, -1.86), p < 0.001 change in % predicted FEV 1 after adjustment for all other potential confounding variables. Systemic inflammation, as measured by hs-CRP, only accounted for a minor mediating pathway effect (9%)., Conclusions: In this homogenous group of dentate men, CP was significantly associated with a reduced respiratory function., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This article was migrated. The article was marked as recommended. Objectives: Intravenous fluid (IV) therapy is an important component of care for many hospital patients, especially in perioperative and acute care settings. However, errors in fluid composition and dosing can be life-threatening. To achieve competent professional performance, i.e., accurate and fluent, it is vitally important that medical students receive effective training in IV fluid therapy. Methods: In this study, we explored how Precision Teaching (PT), a behaviour analytic teaching method, can enhance outcomes of usual medical education techniques. A total of 178 third-year medical students participated in the study during the IV fluid therapy training week. All students completed a multiple-choice test pre- and post-training. In addition to standard IV fluid therapy teaching, the experimental intervention group (n=83 students) used SAFMEDS ( S ay A ll F ast M inute E very D ay S huffled) cards approximately 3-5 times per day for 5 days. The other 95 students (control group) received teaching as usual, but did not undergo the additional training. Results: Results show that the SAFMEDS boosted performance of the intervention group on the MCQ by 20 percentage points when compared to the control group. Fluency (accuracy and speed) of performance on SAFMED trials increased markedly during the intervention week and there was evidence that weaker students benefitted in particular. Conclusions: Implications for medical education are outlined., (Copyright: © 2019 Walsh IK et al.)

In this study, we examined the cellular distribution of encephalopsin (opsin 3; OPN3) expression in the striatum of non-human primates. In addition, because of our long standing interest in Parkinson's disease and neuroprotection, we examined whether parkinsonian (MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) insult and/or photobiomodulation (670 nm) had any impact on encephalopsin expression in this key area of the basal ganglia. Striatal sections of control naïve monkeys, together with those that were either MPTP- and/or photobiomodulation-treated were processed for immunohistochemistry. Our results revealed two populations of striatal interneurones that expressed encephalopsin, one of which was the giant, choline acetyltransferase-containing, cholinergic interneurones. The other population had smaller somata and was not cholinergic. Neither cell group expressed the calcium-binding protein, parvalbumin. There was also rich encephalopsin expression in a set of terminals forming striosome-like patches across the striatum. Finally, we found that neither parkinsonian (MPTP) insult nor photobiomodulation had any effect on encephalopsin expression in the striatum. In summary, our results revealed an extensive network of encephalopsin containing structures throughout the striatum, indicating that external light is in a position to influence a range of striatal activities at both the interneurone and striosome level.

Background: Data from in-vitro, animal, and human lung injury models suggest that keratinocyte growth factor (KGF) might be beneficial in acute respiratory distress syndrome (ARDS). The objective of this trial was to investigate the effect of KGF in patients with ARDS., Methods: We did a double-blind, allocation concealed, randomised, placebo-controlled phase 2 trial in two intensive care units in the UK, involving patients fulfilling the American-European Consensus Conference Definition of ARDS. Patients were randomly assigned (1:1) by computer-generated randomisation schedule with variable block size stratified by site and presence of severe sepsis requiring vasopressors to receive either recombinant human KGF (palifermin 60 μg/kg) or placebo (0·9% sodium chloride solution) daily for a maximum of 6 days. Both patients and investigators were masked to treatment. The primary endpoint was oxygenation index (OI) at day 7. Analyses were by intention to treat. The trial is registered with International Standard Randomised Controlled Trial Registry, number ISRCTN95690673., Findings: Between Feb 23, 2011, and Feb 26, 2014, 368 patients were assessed for eligibility for inclusion in the trial. Of the 60 patients recruited, 29 patients were randomly assigned to receive KGF and 31 to placebo; all were included in the analysis of the primary outcome. There was no significant difference between the two groups in OI at day 7 (mean 62·3 [SD 57·8] in the KGF group, 43·1 [33·5] in the placebo group; mean difference 19·2, 95% CI -5·6 to 44·0, p=0·13). Of interest, although not defined as outcome measures a priori, the KGF group, compared with placebo, had fewer median ventilator-free days (1 day [IQR 0 to 17] in the KGF group vs 20 days [13-22] in the placebo group; difference -8 days, 95% CI -17 to -2; p=0·0002), a longer median duration of ventilation in survivors to day 90 (16 days [IQR 13-30] in the KGF group vs 11 days [8-16] in the placebo group; difference 6 days, 95% CI 2 to 14; p=0·002), and a higher mortality at 28 days (nine [31%] vs three [10%] deaths; risk ratio 3·2, 95% CI 1·0 to 10·7, p=0·054). Adverse events were more frequent in the KGF group than the placebo group (14 vs 5 events; odds ratio 4·9, 95% CI 1·3 to 20·3, p=0·008). The two adverse events assessed as related to KGF were due to pyrexia., Interpretation: KGF did not improve physiological or clinical outcomes in ARDS and might be harmful to patient health., Funding: The Northern Ireland Public Health Agency Research and Development Division., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Aim: To investigate associations between periodontal disease pathogens and levels of systemic inflammation measured by C-reactive protein (CRP)., Methods: A representative sample of dentate 60-70-year-old men in Northern Ireland had a comprehensive periodontal examination. Men taking statins were excluded. Subgingival plaque samples were analysed by quantitative real time PCR to identify the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia. High-sensitivity CRP (mg/l) was measured from fasting blood samples. Multiple linear regression analysis was performed using log-transformed CRP concentration as the dependent variable, with the presence of each periodontal pathogen as predictor variables, with adjustment for various potential confounders., Results: A total of 518 men (mean age 63.6 SD 3.0 years) were included in the analysis. Multiple regression analysis showed that body mass index (p < 0.001), current smoking (p < 0.01), the detectable presence of P. gingivalis (p < 0.01) and hypertension (p = 0.01), were independently associated with an increased CRP. The detectable presence of P. gingivalis was associated with a 20% (95% confidence interval 4-35%) increase in CRP (mg/l) after adjustment for all other predictor variables., Conclusion: In these 60-70-year-old dentate men, the presence of P. gingivalis in subgingival plaque was significantly associated with a raised level of C-reactive protein., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Imaging of human brain as well as cellular and animal models has highlighted a role for the actin cytoskeleton in the development of cell pathology in Alzheimer's disease (AD). Rods and aggregates of the actin-associated protein cofilin are abundant in grey matter of postmortem AD brain and rods are found inside neurites in animal and cell models of AD., Objective: We sought further understanding of the significance of cofilin rods/aggregates to the disease process: Do rods/aggregates correlate with AD progression and the development of hallmark neurofibrillary tangles and neuropil threads? Are cofilin rods/aggregates found in the same neurites as hyperphosphorylated tau?, Methods: The specificity of rods/aggregates to AD compared with general aging and their spatial relationship to tau protein was examined in postmortem human hippocampus, inferior temporal cortex, and anterior cingulate cortex., Results: The presence of cofilin rods/aggregates correlated with the extent of tau pathology independent of patient age. Densities of rods/aggregates were fourfold greater in AD compared with aged-matched control brains and rods/aggregates were significantly larger in AD brain. We did not find evidence for our hypothesis that intracellular cofilin rods are localized to tau-positive neuropil threads. Instead, data suggest the involvement of microglia in the clearance of cofilin rods/aggregates and/or in their synthesis in and around amyloid plaques and surrounding neuropil., Conclusion: Cofilin rods and aggregates signify events initiated early in the pathological cascade. Further definition of the mechanisms leading to their formation in the human brain will provide insights into the cellular causes of AD.

To assess the role of the kynurenine pathway in the pathology of Alzheimer's disease (AD), the expression and localization of key components of the kynurenine pathway including the key regulatory enzyme tryptophan 2,3 dioxygenase (TDO), and the metabolites tryptophan, kynurenine, kynurenic acid, quinolinic acid and picolinic acid were assessed in different brain regions of triple transgenic AD mice. The expression and cell distribution of TDO and quinolinic acid, and their co-localization with neurofibrillary tangles and senile β amyloid deposition were also determined in hippocampal sections from human AD brains. The expression of TDO mRNA was significantly increased in the cerebellum of AD mouse brain. Immunohistochemistry demonstrated that the density of TDO immuno-positive cells was significantly higher in the AD mice. The production of the excitotoxin quinolinic acid strongly increased in the hippocampus in a progressive and age-dependent manner in AD mice. Significantly higher TDO and indoleamine 2,3 dioxygenase 1 immunoreactivity was observed in the hippocampus of AD patients. Furthermore, TDO co-localizes with quinolinic acid, neurofibrillary tangles-tau and amyloid deposits in the hippocampus of AD. These results show that the kynurenine pathway is over-activated in AD mice. This is the first report demonstrating that TDO is highly expressed in the brains of AD mice and in AD patients, suggesting that TDO-mediated activation of the kynurenine pathway could be involved in neurofibrillary tangles formation and associated with senile plaque. Our study adds to the evidence that the kynurenine pathway may play important roles in the neurodegenerative processes of AD.

Purpose: During retinal development, the pattern of blood vessel formation depends upon the combined effects of proliferation and migration of endothelial cells, astrocytes and Müller cells. In this study, we investigated the potential for transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF-2) to influence this process by regulating proliferation and migration of retinal endothelial and macroglial cells., Methods: We assessed the effects of exogenous TGF-β and FGF-2 on the proliferation and migration of cultured endothelial (RF/6A) and Müller cell (MIO-M1) lines. Cell proliferation was measured using a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay over 72 hr. Cell migration was measured using a scratch-wound assay over 72 hr., Results: Transforming growth factor-β inhibited the proliferation of endothelial and Müller cells and inhibited the migration of Müller cells, but not endothelial cells, compared to untreated controls. Conversely, FGF-2 increased endothelial cell proliferation but inhibited endothelial cell migration. Fibroblast growth factor-2 increased migration of Müller cells but had little effect on proliferation except at higher concentrations (20 ng/ml)., Conclusion: Taken together, these observations indicate that TGF-β and FGF could work in concert to inhibit endothelial cell proliferation and migration, respectively; this may have implications for establishing and maintaining the avascular zone of primate fovea., (© 2010 The Authors. Journal compilation © 2010 Acta Ophthalmol.)

Background: Certain pharmacological agents administered during electroconvulsive therapy may have the potential to prevent persistent retrograde amnesia induced during electroconvulsive therapy. This review examines mechanisms for electroconvulsive therapy-induced retrograde amnesia, and evaluates the suitability of the anaesthetic ketamine for preventing this amnestic outcome., Methods: A review of human studies, animal models and theoretical models in light of memory dysfunction following electroconvulsive therapy was conducted. MEDLINE was searched from 1950 to April 2009 using the MeSH terms "electroconvulsive therapy", "memory", "memory short term", "memory disorders", "excitatory amino acid antagonists", and "ketamine". PREMEDLINE was searched using the terms "electroconvulsive therapy", "amnesia" and "ketamine". Additional keyword and reference list searches were performed. No language, date constraints or article type constraints were used., Results: Disruption of long term potentiation as a mechanism for electroconvulsive therapy-induced retrograde amnesia is well supported. Based on this putative mechanism, an N-methyl-D-aspartate receptor antagonist would appear suitable for preventing the retrograde amnesia. Available evidence in animals and humans supports the prediction that ketamine, an anaesthetic agent and N-methyl-D-aspartate receptor antagonist, could effectively prevent electroconvulsive therapy-induced persistent retrograde amnesia. Whilst there are concerns about the use of ketamine with electroconvulsive therapy, such as possible psychotomimetic effects, on balance this anaesthetic agent may improve or hasten clinical response to electroconvulsive therapy., Conclusions: A clinical trial is warranted to determine if ketamine anaesthesia during electroconvulsive therapy can lessen persistent retrograde amnesia and improve therapeutic response. Electroconvulsive therapy with ketamine anaesthesia may provide effective antidepressant action with minimal side effects., (Copyright 2009 Elsevier B.V. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease of unknown pathogenesis. The kynurenine pathway (KP), activated during neuroinflammation, is emerging as a possible contributory factor in ALS. The KP is the major route for tryptophan (TRP) catabolism. The intermediates generated can be either neurotoxic, such as quinolinic acid (QUIN), or neuroprotective, such as picolinic acid (PIC), an important endogenous chelator. The first and inducible enzyme of the pathway is indoleamine 2,3-dioxygenase (IDO). The present study aimed to characterize the expression of the KP in cerebrospinal fluid (CSF), serum and central nervous system (CNS) tissue of ALS patients. Using high performance liquid chromatography, we analysed the levels of TRP and kynurenine (KYN), and, with gas chromatography/mass spectrometry, the levels of PIC and QUIN, in the CSF and serum of ALS patients and control subjects. Immunohistochemistry was employed to determine the expression of QUIN, IDO and human leukocyte antigen-DR (HLA-DR) in sections of brain and spinal cord from ALS patients. There were significantly increased levels of CSF and serum TRP (P < 0.0001), KYN (P < 0.0001) and QUIN (P < 0.05) and decreased levels of serum PIC (P < 0.05) in ALS samples. There was a significant increase in activated microglia expressing HLA-DR (P < 0.0001) and increased neuronal and microglial expression of IDO and QUIN in ALS motor cortex and spinal cord. We show the presence of neuroinflammation in ALS and provide the first strong evidence for the involvement of the KP in ALS. These data point to an inflammation-driven excitotoxic-chelation defective mechanism in ALS, which may be amenable to inhibitors of the KP.

In Alzheimer's disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads), and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that, during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence resonance energy transfer analysis revealed colocalization of cofilin-GFP (green fluorescent protein) and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and small interfering RNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally, we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous amyloid-beta (Abeta) peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction, or Abeta peptides, all suspected initiators of synaptic loss and neurodegeneration in AD.

Some of the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QA), are likely to play a role in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the KP is over activated in AD brain and that QA accumulates in amyloid plaques and within dystrophic neurons. We hypothesized that QA in pathophysiological concentrations affects tau phosphorylation. Using immunohistochemistry, we found that QA is co-localized with hyperphosphorylated tau (HPT) within cortical neurons in AD brain. We then investigated in vitro the effects of QA at various pathophysiological concentrations on tau phosphorylation in primary cultures of human neurons. Using western blot, we found that QA treatment increased the phosphorylation of tau at serine 199/202, threonine 231 and serine 396/404 in a dose dependent manner. Increased accumulation of phosphorylated tau was also confirmed by immunocytochemistry. This increase in tau phosphorylation was paralleled by a substantial decrease in the total protein phosphatase activity. A substantial decrease in PP2A expression and modest decrease in PP1 expression were observed in neuronal cultures treated with QA. These data clearly demonstrate that QA can induce tau phosphorylation at residues present in the PHF in the AD brain. To induce tau phosphorylation, QA appears to act through NMDA receptor activation similar to other agonists, glutamate and NMDA. The QA effect was abrogated by the NMDA receptor antagonist memantine. Using PCR arrays, we found that QA significantly induces 10 genes in human neurons all known to be associated with AD pathology. Of these 10 genes, 6 belong to pathways involved in tau phosphorylation and 4 of them in neuroprotection. Altogether these results indicate a likely role of QA in the AD pathology through promotion of tau phosphorylation. Understanding the mechanism of the neurotoxic effects of QA is essential in developing novel therapeutic strategies for AD.

Here we show a new effect of Ca2+ on microtubule morphology: Ca2+ can cause smooth curving of microtubules in the presence of microtubule-associated proteins (MAPs). In vitro, microtubules self-organize, forming complex dissipative structures. Such structures may be strongly affected by relatively weak external factors. A factor such as Ca2+ potentially influences spatiotemporal patterns of microtubule assembly, but the dynamics are unclear. We tested Ca2+ effects on microtubule formation. Using EM, microtubule length, curvature, and alignment and were measured in two systems: 2 mg/ml microtubule protein containing MAPs and 1 mM EGTA with and without 1 mM Ca2+. The two systems were then tested using light scattering. In low Ca2+, a birefringent microtubular pattern is seen, increasing with polymerization. When 1 mM Ca2+ is added to the solution, anisotropic phase is prevented without microtubule disruption. This demonstrates an additional mechanism by which Ca2+ can alter the dynamics and morphology of microtubules.

The kynurenine pathway is a major route of L-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

Amyloid-rich plaques are a feature of the aging human cerebral cortex. We have recently described another feature of aging human cortex, microhaemorrhages, identified by their content of haem, red blood cells, collagen and clotting factors, and their spatial relationship to capillaries. Here we relate microhaemorrhages to amyloid deposits. Observations were made in three groups: patients with no history of dementia, patients with Alzheimer's disease (AD) and patients with Down's syndrome (DS) and dementia. Amyloid deposits and microhaemorrhages were labelled in adjacent sections, amyloid deposits with antibodies to beta-amyloid (betaA), and microhaemorrhages by Prussian blue histochemistry for haem. The densities and sizes of betaA deposits and haem-rich deposits (HRDs), and their relationship to blood vessels, were surveyed in temporal, cingulate and superior frontal cortex. Our results suggest that HRDs and betaA deposits are the same sites of pathology. Their densities in the cortex and white matter of the regions surveyed varied markedly between cases, particularly between demented and non-demented cases, but they always co-varied; where haem deposits were sparse or numerous, so were betaA deposits. Both HRDs and betaA deposits formed adjacent to or encircling small vessels, often at branch points, and a spatial proximity analysis confirmed that both were found close to or colocalising with microvessels. Both HRDs and betaA deposits were associated with blood- or vessel-derived proteins (fibrinogen, von Willebrand factor and collagen VI). Since haem is an established marker of cerebral bleeding, and amyloid is a marker of senile plaques, our results indicate that senile plaques are sites of microhaemorrhages. This colocalisation raises the very testable questions of whether microhaemorrhages are early events in plaque formation and whether therapies which stabilise cerebral microvessels can prevent the onset or slow the progress of dementias associated with plaque formation.

In this post mortem study, we examined haem-rich deposits (HRDs) in patients with and without dementia, using a histochemical label (Prussian blue) to show haem, autofluorescence to detect red blood cells (RBCs), and immunohistochemistry for clotting-related factors and collagen IV. The patients studied had no clinical or post mortem evidence of macrovascular stroke. To allow examination of the spatial relationships between HRDs and the microvasculature, we cut 45-microm sections. Haem-rich deposits were small (<200 microm diameter). They were rare in younger (<50 years) patients but were more common in older (>70 years) patients, particularly in cerebral cortex, and were most abundant in cases with senile plaques. Wherever HRDs appeared they were perivascular and appeared to form around capillaries or small arterioles. Using a software package (Proxan) developed to outline vessels and HRDs, and to analyse the distances between them, a tight spatial correlation between HRDs and capillaries was shown. In addition, HRDs were rich in von Willebrand factor (vWF), fibrinogen, collagen IV and RBCs. These observations suggest that HRDs are the residua of capillary bleeds (microhaemorrhages), and that microhaemorrhages are a common feature of the aging cerebral cortex, particularly where plaque pathology is present.

The toxicology of metallic mercury on motor neurons and their processes requires further work to resolve controversial implications in the aetiology of human motor neuron disease (MND). The assessment of experimental neurotoxicity in the peripheral motor system is, however, technically problematic and difficult to interpret. The mean number of axons in a nerve can vary considerably due to a high degree of biological variation. Atrophy of large axons can appear as loss when, in fact, their numbers appear in smaller diameter axonal categories. We addressed these quantitative problems using the murine phrenic nerve (MPN), a mono-fascicular, predominantly motor nerve as a model system. One micrometer transverse sections of gluteraldehyde/osmium tetroxide fixed MPNs were stained for myelin using a silver technique. Axon areas were measured from digital images of the nerve in cross-section (ImagePro Plus software) and transformed to circular diameter equivalents, then displayed as frequency distributions. We found a high biological variation in the mean axon number between paired nerves within experimental groups. Therefore, axon diameter data within individuals group was pooled. Theoretical simulation of axonal degeneration, atrophy and hypertrophy of larger myelinated axons (also affected in MND) were modelled by manipulating the original data set. With this model, by comparing normal distributions, it is possible to distinguish axonal atrophy, degenerative loss, and hypertrophy as distinct pathological processes in the large calibre axon subgroup that are selectively vulnerable to metallic toxins such as mercury.

The present immunohistochemical study provides evidence that the kynurenine pathway is up-regulated in Alzheimer's disease (AD) brain, leading to increases in the excitotoxin quinolinic acid (QUIN). We show that the regulatory enzyme of the pathway leading to QUIN synthesis, indoleamine 2,3 dioxygenase (IDO) is abundant in AD compared with controls. In AD hippocampus, both IDO- and QUIN-immunoreactivity (-IR) was detected in cortical microglia, astrocytes and neurones, with microglial and astrocytic expression of IDO and QUIN highest in the perimeter of senile plaques. QUIN-IR was present in granular deposits within the neuronal soma of AD cortex and was also seen uniformly labelling neurofibrillary tangles. Our data imply that QUIN may be involved in the complex and multifactorial cascade leading to neuro-degeneration in AD. These results may open a new therapeutic door for AD patients.

Neurofibrillary tangle staging was compared in the nucleus basalis and cerebral cortex of Alzheimer's disease patients with and without Lewy body disease. In pure Alzheimer's disease, cholinergic nucleus basalis cell number, as determined from counts in serial forebrain sections, was 22-60% of control mean, with the majority of residual cells containing tangles. A comparison between control cell number and the combined number of tangles plus tangle-free neurons in pure Alzheimer's disease suggests that the majority of nucleus basalis neurons were lost through neurofibrillary degeneration. The staging of neurofibrillary degeneration in the nucleus basalis was discordant with cortical changes as some controls had more extensive tangle formation in the nucleus basalis than in the cerebral cortex. Patients having both Alzheimer's disease and Lewy body pathology had few or no tangles in the nucleus basalis despite greater loss of neurons than purely demented patients. The presence of concomitant pathology had a greater effect on nucleus basalis tangle burden than did cortical disease stage, suggesting dichotomous disease processes in the cerebral cortex and forebrain.

Background: The cholinergic nucleus basalis (Ch4) is an exclusive site of neurofibrillary degeneration in alcoholic patients with Wernicke's encephalopathy., Aim: To test the hypothesis that the loss of Ch4 neurons contributes to the memory disorder, Korsakoff's psychosis, commonly seen in Wernicke's encephalopathy., Methods: Magnocellular basal forebrain neurons were quantified in alcoholic patients with Wernicke's encephalopathy, both with and without Korsakoff's psychosis, and neurologically asymptomatic alcoholic and non-alcoholic controls. Because amnesic and non-amnesic patients with Wernicke's encephalopathy share common periventricular lesions, both thiamine deficient groups as well as alcoholic patients with no neurological complications were included to determine the lesion specific to memory impairment., Results: Ch4 cell number did not differ significantly between alcoholic and non-alcoholic controls and there was no correlation between cell number and lifetime alcohol intake. However, Ch4 cell number in all groups was significantly correlated with the volume of its major projection target, the cerebral cortex. Ch4 cell number in the non-amnesic Wernicke's encephalopathy group was significantly below controls (24%), with cell number in patients with Korsakoff's psychosis 21% below controls. There was considerable overlap in cell number between groups. On discriminant analysis, there was significantly greater cell loss in three non-amnesic patients with Wernicke's encephalopathy than in some patients with Korsakoff's psychosis. The nonamnesic patient with the greatest cell loss was impaired on attentional tasks., Conclusion: Whereas neurons in the nucleus basalis are at risk in thiamine deficient alcoholic patients, cell loss is minor and does not account for the profound memory disorder.

Cortical atrophy and cell loss in the cholinergic nucleus basalis is a well-established characteristic of Alzheimer's disease; however, previous studies not have analysed cholinergic cell loss and cortical atrophy in concert. In autopsy brains from eight patients with Alzheimer's disease and 12 control subjects, the numbers of nucleus basalis neurons were determined from 50-microm serial Nissl-stained sections. Volumes of the cerebrum, cortical gray matter (total, lobar and subregional), white matter and deep gray structures were computed by point counting on black and white photographs of gapless 3-mm coronal slices of formalin-fixed brains. Cell loss in the nucleus basalis was found to range between 89% and 42% in Alzheimer's disease compared with controls. White matter volume was unchanged in absolute terms in Alzheimer's disease patients compared with controls, while cortical volume was significantly reduced. Gray matter atrophy was most prominent in temporal and frontal cortices. A highly significant linear relationship was found between cortical volume and nucleus basalis cell number in controls and Alzheimer's disease patients, with values for both groups on a single regression line. Whole brain and cerebral volumes were also highly correlated to nucleus basalis cell numbers in both groups. A quantitative analysis of plaque and tangle burden in cortical target areas of the nucleus basalis was performed. In contrast to the relationship with cortical volume, nucleus basalis cell number and neurofibrillary tangle number were not significantly correlated to the density of cortical histopathology. These results suggest that the volume of cortical gray matter is coupled to the number of nucleus basalis neurons. Compromised viability of nucleus basalis neurons may precede cortical volume loss as large numbers of neurofibrillary tangles, detected with nickel peroxidase staining, were found in this nucleus in all Alzheimer's disease cases, including those with minimal cell loss.

Stain sensitivity is a key factor in estimating the frequency of plaques and neurofibrillary tangles in Alzheimer's disease (AD), making it essential that the sensitivity and selectivity of detection methods for identifying these lesions is maximized. Several new, improved techniques have recently been described, although these methods have not been compared quantitatively with those techniques currently recommended for use in standardized diagnostic protocols. In the present study, eight different stains were examined for their selectivity and sensitivity in detecting plaques and tangles in serial tissue sections from AD and control brains. Techniques compared were immunohistochemistry for tau and beta-amyloid, thioflavin S, nickel peroxidase method, and four silver impregnation techniques (Gallyas silver iodide, Campbell-Switzer-Martin, Garvey's modified Bielschowsky and methenamine silver methods). Among these eight staining techniques, the nickel peroxidase proved the most reliable method for the demonstration of the histopathological lesions of AD. This method labels all morphological types of plaques and tangles within a single tissue section, and provides several advantages for the analysis of lesion progression and distribution.

Glial fibrillary acidic protein (GFAP) is the principal marker for brain astrocytes. The present study aims to examine the variability in GFAP immunohistochemistry in formalin-fixed human brain. Four commercially-available antisera were tested using standardised protocols in the cerebral cortex of three cases with prominent glial reactions and one control. GFAP immunoreactivity was largely confined to the pial surface and white matter in control cortex, with the number of astrocytic cell bodies and processes as well as intensity of staining markedly increased in damaged cortices. A dramatic difference in the pattern of GFAP staining using different antisera was observed and may account for discrepancies between past studies. This variance has important practical implications for the interpretation of results using GFAP immunohistochemistry in human tissue.

Magnocellular neurons in the cholinergic nucleus basalis appear to be vulnerable in a variety of pathological conditions, including chronic alcoholism. While neurofibrillary degeneration of these neurons has been noted in a number of disorders characterized by dementia, the mechanism of cell death in thiamine-deficient chronic alcoholics has not been identified. In the present post-mortem investigation, multiple brain regions of seven thiamine-deficient chronic alcoholics, three neurologically asymptomatic chronic alcoholics and seven non-alcoholic age matched controls were screened for neurofibrillary pathology using both tau-immunohistochemistry and a modified Bielschowsky silver stain. In chronic alcoholics with thiamine deficiency, neurofibrillary pathology was found in the nucleus basalis, but not any other brain region. Neurofibrillary tangles were not seen in age-matched controls and were infrequent in alcoholics without neuropathological signs of thiamine-deficiency. Neurofibrillary tangles were most numerous in those cases with cell loss in the nucleus basalis. These findings suggest that neurodegeneration of the nucleus basalis in chronic alcoholics proceeds through the formation of neurofibrillary tangles.

Tau immunoreactivity was examined in post mortem tissue from patients in three groups: neurologically-asymptomatic and neuropathologically normal alcoholics, alcoholics with Wernicke's Encephalopathy (WE) and age matched non-alcoholic controls. Tau-positive granular and fibrillary inclusions were frequently observed within the magnocellular neurons of the cholinergic nucleus basalis, within occasional nucleus basalis neurons in non-WE alcoholics, but not in controls. Tau immunoreactivity was not however observed in cortical, brainstem, diencephalic or non-cholinergic forebrain structures. Peroxidase activity was also examined within the nucleus basalis using diaminobenzidine as an indicator. The majority of neurons in the basal forebrain showed increased peroxidase activity in all WE alcoholics and in some nucleus basalis neurons of non-WE alcoholics, but was rarely seen in controls. Neighboring astrocytes also showed increased peroxidase activity. These results suggest a link between peroxidase activity and the abnormal accumulation of phosphorylated tau. The presence of tau in the nucleus basalis of alcoholics with WE suggests a thiamine-dependent mechanism in tau accumulation and cell death in the cholinergic basal forebrain.

A reliable new method was developed for detecting neurofibrillary tangles, senile plaques, and neuropil threads in human neural tissue. Excellent morphological definition of the pathological structures was achieved with this procedure without staining normal neuronal and glial elements. The technique was applied to cortical tissue from eight patients with Alzheimer's disease and three controls. Histological sections from these cases were incubated in an avidin-biotin-peroxidase complex solution containing 0.5% nickel ammonium sulfate, followed by visualization in 3,3'-diaminobenzidine and H2O2. Although the avidin-biotin system is routinely used in immunohistochemistry, no antibodies were employed in the present procedure. This technique has advantages over silver impregnation methods because it requires very little monitoring of critical steps and yields superior results. The method is suitable for processing large numbers of tissue sections per staining run, and the results are highly reproducible. These features are advantageous in research studies comparing the distribution of lesions in large numbers of cases. The precise mechanism of staining has not been determined; however, conditions such as nickel concentrations, pH, and avidin-biotin-peroxidase complex concentrations were varied to examine critical steps in the process.

We have analyzed glial changes in forebrain and diencephalic regions in 19 alcoholics with different histories of chronic alcohol consumption and related medical complications including Wernicke's encephalopathy and alcoholic liver disease. Cases with postmortem evidence of hepatic encephalopathy were excluded. Two of the alcoholic patients had ceased drinking for several years prior to death. Brains were obtained postmortem and fixed in formalin. Serial 50 microns sections of the forebrain and diencephalon at 750 microns intervals were stained with standard histochemical stains (haematoxylin and eosin, luxol fast blue, cresyl violet and silver), as well as immunohistochemically for glial fibrillary acidic protein (GFAP). In control tissue, GFAP-positive astrocytes were intimately associated with ependymal, pial, and vascular surfaces. In alcoholic cases, the morphology of these cells was markedly changed showing enlargement of the cell bodies and beading of the cellular processes. In contrast to controls, GFAP-positive astrocytes were seen within and surrounding clusters of magnocellular neurons in the basal forebrain and hypothalamus. In thiamine-deficient alcoholics, glial scarring in the vicinity of the large branches of the cerebral arteries disrupted the normal forebrain architecture. A patchy loss of GFAP immunostaining was seen in most severe cases. A remarkable number of corpora amylacea also rimmed blood vessels, pial and ependymal surfaces in all alcoholics compared to controls. The beaded fibers were seen in alcoholics drinking at the time of death as well as in those who had ceased drinking alcohol several years prior to death. These results indicate that chronic alcoholics have prominent glial changes which persist despite the cessation of alcohol consumption and are not exclusive to alcoholics with liver pathology.

The decreasing cost and increasing availability of computers have created both concern and desire on the part of the non-computer professional. This article provides basic information to use in the selection of microcomputer hardware and software. It is slanted toward the small practitioner, one to four physicians, and provides practical guidelines, definitions, and examples relevant for a small practice.

Contemporary research perspectives on the impact of unemployment on health and well-being among young people have tended to focus on a rather narrow range of outcomes, typically those in the mental health domain. The impetus for the proposed longitudinal study, the main dimensions of which are described in this paper, reflects the need for a more comprehensive profiling of the health needs and experiences of young people if effective interventions tailored to their short and long term health needs are to be developed. The proposed study includes variables from a wide range of domains and adopts an interdisciplinary perspective. The feasibility of the approach, both in terms of establishing appropriately stratified samples and determining the acceptability and utility of the measures proposed has been examined during an extensive pilot phase. Findings from the database established during this phase are presented. These focus on multi-dimensional comparisons of health and well-being between employed and unemployed young people, the impact of socio-economic status of origin on cardiovascular and other indicators, and the correlates of health and well-being among the unemployed. The results point to the potential complexity of the influences on health status and behaviour and the need to develop comprehensive models of this for research and intervention purposes.