Expression microdissection isolation of enriched cell populations from archival brain tissue

BACKGROUND: Laser capture microdissection (LCM) is an established technique for the procurement of enriched cell populations that can undergo further downstream analysis, although it does have limitations. Expression microdissection (xMD) is a new technique that begins to address these pitfalls, such as operator dependence and contamination. NEW METHOD: xMD utilises immunohistochemistry in conjunction with a chromogen to isolate specific cell types by extending the fundamental principles of LCM to create an operator-independent method for the procurement of specific CNS cell types. RESULTS: We report how xMD enables the isolation of specific cell populations, namely neurones and astrocytes, from rat formalin fixed-paraffin embedded (FFPE) tissue. Subsequent reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirms the enrichment of these specific populations. RIN values after xMD indicate samples are sufficient to carry out further analysis. COMPARISON WITH EXISTING METHOD: xMD offers a rapid method of isolating specific CNS cell types without the need for identification by an operator, reducing the amount of unintentional contamination caused by operator error, whilst also significantly reducing the time required by the current basic LCM technique. CONCLUSIONS: xMD is a superior method for the procurement of enriched cell populations from post-mortem tissue, which can be utilised to create transcriptome profiles, aiding our understanding of the contribution of these cells to a range of neurological diseases. xMD also addresses the issues associated with LCM, such as reliance on an operator to identify target cells, which can cause contamination, as well as addressing the time consuming nature of LCM

TDP43 proteinopathy is associated with aberrant DNA methylation in human amyotrophic lateral sclerosis

Background Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neurone (MN) degeneration and death. ALS can be sporadic (sALS) or familial, with a number of associated gene mutations, including C9orf72 (C9ALS). DNA methylation is an epigenetic mechanism whereby a methyl group is attached to a cytosine (5mC), resulting in gene expression repression. 5mC can be further oxidized to 5‐hydroxymethylcytosine (5hmC). DNA methylation has been studied in other neurodegenerative diseases, but little work has been conducted in ALS. Aims To assess differences in DNA methylation in individuals with ALS and the relationship between DNA methylation and TDP43 pathology. Methods Post mortem tissue from controls, sALS cases and C9ALS cases were assessed by immunohistochemistry for 5mC and 5hmC in spinal cord, motor cortex and prefrontal cortex. LMNs were extracted from a subset of cases using laser capture microdissection. DNA from these underwent analysis using the MethylationEPIC array to determine which molecular processes were most affected. Results There were higher levels of 5mC and 5hmC in sALS and C9ALS in the residual lower motor neurones (LMNs) of the spinal cord. Importantly, in LMNs with TDP43 pathology there was less nuclear 5mC and 5hmC compared to the majority of residual LMNs that lacked TDP43 pathology. Enrichment analysis of the array data suggested RNA metabolism was particularly affected. Conclusions DNA methylation is a contributory factor in ALS LMN pathology. This is not so for glia or neocortical neurones

Establishing mRNA and microRNA interactions driving disease heterogeneity in amyotrophic lateral sclerosis patient survival

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease, associated with the degeneration of both upper and lower motor neurons of the motor cortex, brainstem and spinal cord. Death in most patients results from respiratory failure within 3–4 years from symptom onset. However, due to disease heterogeneity some individuals survive only months from symptom onset while others live for several years. Identifying specific biomarkers that aid in establishing disease prognosis, particularly in terms of predicting disease progression, will help our understanding of amyotrophic lateral sclerosis pathophysiology and could be used to monitor a patient’s response to drugs and therapeutic agents. Transcriptomic profiling technologies are continually evolving, enabling us to identify key gene changes in biological processes associated with disease. MicroRNAs are small non-coding RNAs typically associated with regulating gene expression, by degrading mRNA or reducing levels of gene expression. Being able to associate gene expression changes with corresponding microRNA changes would help to distinguish a more complex biomarker signature enabling us to address key challenges associated with complex diseases such as amyotrophic lateral sclerosis. The present study aimed to investigate the transcriptomic profile (mRNA and microRNA) of lymphoblastoid cell lines from amyotrophic lateral sclerosis patients to identify key signatures that are distinguishable in those patients who suffered a short disease duration (6 years) (n = 20). Transcriptional profiling of microRNA–mRNA interactions from lymphoblastoid cell lines in amyotrophic lateral sclerosis patients revealed differential expression of genes involved in cell cycle, DNA damage and RNA processing in patients with longer survival from disease onset compared with those with short survival. Understanding these particular microRNA–mRNA interactions and the pathways in which they are involved may help to distinguish potential therapeutic targets that could exert neuroprotective effects to prolong the life expectancy of amyotrophic lateral sclerosis patients

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Azithromycin in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatory actions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19. Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospital with COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients were randomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once per day by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatment groups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment and were twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants and local study staff were not masked to the allocated treatment, but all others involved in the trial were masked to the outcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) were eligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was 65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomly allocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall, 561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days (rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median 10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days (rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, no significant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilation or death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24). Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or other prespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restricted to patients in whom there is a clear antimicrobial indication. Funding UK Research and Innovation (Medical Research Council) and National Institute of Health Research