Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system.

Arginine methylation is a post-translational modification that consists of the transfer of one or two methyl (CH3) groups to arginine residues in proteins. Several types of arginine methylation occur, namely monomethylation, symmetric dimethylation and asymmetric dimethylation, which are catalysed by different protein arginine methyltransferases (PRMTs). Inhibitors of PRMTs have recently entered clinical trials to target several types of cancer, including gliomas (NCT04089449). People with glioblastoma (GBM), the most aggressive form of brain tumour, are among those with the poorest quality of life and likelihood of survival of anyone diagnosed with cancer. There is currently a lack of (pre)clinical research on the possible application of PRMT inhibitors to target brain tumours. Here, we set out to investigate the effects of clinically-relevant PRMT inhibitors on GBM biopsies. We present a new, low-cost, easy to fabricate perfusion device that can maintain GBM tissue in a viable condition for at least eight days post-surgical resection. The miniaturised perfusion device enables the treatment of GBM tissue with PRMT inhibitors ex vivo, and we observed a two-fold increase in apoptosis in treated samples compared to parallel control experiments. Mechanistically, we show thousands of differentially expressed genes after treatment, and changes in the type of arginine methylation of the RNA binding protein FUS that are consistent with hundreds of differential gene splicing events. This is the first time that cross-talk between different types of arginine methylation has been observed in clinical samples after treatment with PRMT inhibitors.

An external ventricular drainage catheter impregnated with rifampicin, trimethoprim and triclosan, with extended activity against MDR Gram-negative bacteria: an in vitro and in vivo study.

BACKGROUND: External ventricular drainage (EVD) carries a high risk of ventriculitis, increasingly caused by MDR Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii. Existing antimicrobial EVD catheters are not effective against these, and we have developed a catheter with activity against MDR bacteria and demonstrated the safety of the new formulation for use in the brain. OBJECTIVES: Our aim was to determine the ability of a newly formulated impregnated EVD catheters to withstand challenge with MDR Gram-negative bacteria and to obtain information about its safety for use in the CNS. METHODS: Catheters impregnated with three antimicrobials (rifampicin, trimethoprim and triclosan) were challenged in flow conditions at four weekly timepoints with high doses of MDR bacteria, including MRSA and Acinetobacter, and monitored for bacterial colonization. Catheter segments were also inserted intracerebrally into Wistar rats, which were monitored for clinical and behavioural change, and weight loss. Brains were removed after either 1 week or 4 weeks, and examined for evidence of inflammation and toxicity. RESULTS: Control catheters colonized quickly after the first challenge, while no colonization occurred in the impregnated catheters even after the 4 week challenge. Animals receiving the antimicrobial segments behaved normally and gained weight as expected. Neurohistochemistry revealed only surgical trauma and no evidence of neurotoxicity. CONCLUSIONS: The antimicrobial catheter appears to withstand bacterial challenge for at least 4 weeks, suggesting that it might offer protection against infection with MDR Gram-negative bacteria in patients undergoing EVD. It also appears to be safe for use in the CNS.

An unusual pituitary adenoma coexistent with bilateral meningiomas: case report.

The association of pituitary adenomas and meningioma is rare. We present the case of a 46 year old lady who initially presented with large bilateral meningiomas and acromegaly. Histology demonstrated mammosomatotroph cell adenoma co-expressing Growth Hormone. This appears to be the first description of mammosomatotroph cell adenoma associated with meningiomas in the literature.

Tissue metabolite profiles for the characterisation of paediatric cerebellar tumours.

Paediatric brain tumors are becoming well characterized due to large genomic and epigenomic studies. Metabolomics is a powerful analytical approach aiding in the characterization of tumors. This study shows that common cerebellar tumors have metabolite profiles sufficiently different to build accurate, robust diagnostic classifiers, and that the metabolite profiles can be used to assess differences in metabolism between the tumors. Tissue metabolite profiles were obtained from cerebellar ependymoma (n = 18), medulloblastoma (n = 36), pilocytic astrocytoma (n = 24) and atypical teratoid/rhabdoid tumors (n = 5) samples using HR-MAS. Quantified metabolites accurately discriminated the tumors; classification accuracies were 94% for ependymoma and medulloblastoma and 92% for pilocytic astrocytoma. Using current intraoperative examination the diagnostic accuracy was 72% for ependymoma, 90% for medulloblastoma and 89% for pilocytic astrocytoma. Elevated myo-inositol was characteristic of ependymoma whilst high taurine, phosphocholine and glycine distinguished medulloblastoma. Glutamine, hypotaurine and N-acetylaspartate (NAA) were increased in pilocytic astrocytoma. High lipids, phosphocholine and glutathione were important for separating ATRTs from medulloblastomas. This study demonstrates the ability of metabolic profiling by HR-MAS on small biopsy tissue samples to characterize these tumors. Analysis of tissue metabolite profiles has advantages in terms of minimal tissue pre-processing, short data acquisition time giving the potential to be used as part of a rapid diagnostic work-up.

Histological Basis of Laminar MRI Patterns in High Resolution Images of Fixed Human Auditory Cortex.

Functional magnetic resonance imaging (fMRI) studies of the auditory region of the temporal lobe would benefit from the availability of image contrast that allowed direct identification of the primary auditory cortex, as this region cannot be accurately located using gyral landmarks alone. Previous work has suggested that the primary area can be identified in magnetic resonance (MR) images because of its relatively high myelin content. However, MR images are also affected by the iron content of the tissue and in this study we sought to confirm that different MR image contrasts did correlate with the myelin content in the gray matter and were not primarily affected by iron content as is the case in the primary visual and somatosensory areas. By imaging blocks of fixed post-mortem cortex in a 7 T scanner and then sectioning them for histological staining we sought to assess the relative contribution of myelin and iron to the gray matter contrast in the auditory region. Evaluating the image contrast in [Formula: see text]-weighted images and quantitative [Formula: see text] maps showed a reasonably high correlation between the myelin density of the gray matter and the intensity of the MR images. The correlation with T1-weighted phase sensitive inversion recovery (PSIR) images was better than with the previous two image types, and there were clearly differentiated borders between adjacent cortical areas in these images. A significant amount of iron was present in the auditory region, but did not seem to contribute to the laminar pattern of the cortical gray matter in MR images. Similar levels of iron were present in the gray and white matter and although iron was present in fibers within the gray matter, these fibers were fairly uniformly distributed across the cortex. Thus, we conclude that T1- and [Formula: see text]-weighted imaging sequences do demonstrate the relatively high myelin levels that are characteristic of the deep layers in primary auditory cortex and allow it and some of the surrounding areas to be reliably distinguished.

Dissecting DNA repair in adult high grade gliomas for patient stratification in the post-genomic era.

Deregulation of multiple DNA repair pathways may contribute to aggressive biology and therapy resistance in gliomas. We evaluated transcript levels of 157 genes involved in DNA repair in an adult glioblastoma Test set (n=191) and validated in 'The Cancer Genome Atlas' (TCGA) cohort (n=508). A DNA repair prognostic index model was generated. Artificial neural network analysis (ANN) was conducted to investigate global gene interactions. Protein expression by immunohistochemistry was conducted in 61 tumours. A fourteen DNA repair gene expression panel was associated with poor survival in Test and TCGA cohorts. A Cox multivariate model revealed APE1, NBN, PMS2, MGMT and PTEN as independently associated with poor prognosis. A DNA repair prognostic index incorporating APE1, NBN, PMS2, MGMT and PTEN stratified patients in to three prognostic sub-groups with worsening survival. APE1, NBN, PMS2, MGMT and PTEN also have predictive significance in patients who received chemotherapy and/or radiotherapy. ANN analysis of APE1, NBN, PMS2, MGMT and PTEN revealed interactions with genes involved in transcription, hypoxia and metabolic regulation. At the protein level, low APE1 (p=0.031) and low PTEN (p=0.042) remain associated with poor prognosis. In conclusion, multiple DNA repair pathways operate to influence biology and clinical outcomes in adult high grade gliomas.

The ubiquitin-binding protein p62 identifies argyrophilic grain pathology with greater sensitivity than conventional silver stains.

The routine diagnosis of argyrophilic grain disease is fraught by the lack of availability of an easily applied reproducible stain that can highlight the grain pathology with sensitivity and with minimal background. The Gallyas silver iodide technique is not widely used and, even in experienced hands, is difficult to perform due to inconsistencies inherent in silver-based techniques on thin sections. Grain pathology can be detected using immunohistochemistry for phosphorylated tau protein, but the grain pathology is most often masked by background tau-positive material; leading to problems with interpretation, especially for practitioners seeing small numbers of cases. There is a need for a reliable immunohistochemical stain that can detect grain pathology and provide a clear contrast between grains and other tau-positive neurodegenerative pathologies. We have investigated the novel ubiquitin-binding protein p62 as a potential biomarker for grain pathology in argyrophilic grain disease. Four cases of argyrophilic grain disease, in which the pathology was determined using the Gallyas silver iodide technique, were re-assessed using paraffin-embedded sections immunostained with antibodies specific for p62. We found that the detection of grain pathology was more sensitive than with silver-based techniques and that the resolution of the pathology was significantly improved. We suggest that p62 could be used to replace the Gallyas technique in the routine diagnosis of argyrophilic grain disease.

Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: applications in diagnostic neuropathology.

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.

Hepatocyte expression of minichromosome maintenance protein-2 predicts fibrosis progression after transplantation for chronic hepatitis C virus: a pilot study.

Although graft infection with hepatitis C virus (HCV) occurs in virtually all patients transplanted for HCV-related liver disease, the outcome ranges from minimal disease to the rapid development of cirrhosis. Induction of hepatocyte cell cycle entry followed by inhibition of cell cycle progression has been proposed as a potential mechanism whereby HCV may cause hepatocyte dysfunction and may promote fibrogenesis. The aim of this study was to assess whether early hepatocyte cell cycle entry might predict subsequent fibrosis progression in patients with graft HCV infection after liver transplantation. Liver biopsies from 21 liver transplant recipients diagnostic of graft HCV infection but before development of significant fibrosis were studied. Patients were classed as nonprogressors, intermediate progressors, or rapid progressors according to the rate of fibrosis progression calculated from the most recent biopsy. Minichromosome maintenance protein 2 (Mcm-2), a highly sensitive and specific marker of cell cycle entry, and cyclin-dependent kinase inhibitor p21 were detected by immunohistochemistry. Hepatocyte Mcm-2 expression increased significantly according to rate of fibrosis. For nonprogressors, the median percentage of positive hepatocytes was 5.3% (range, 0.92%-11.2%) compared with 20.7% (4.6%-43.7%) in intermediate progressors and 23.7% (11.6%-55.2%) in rapid progressors (P = 0.002). By contrast, there was no evidence of a difference in hepatocyte p21 expression. Median values and ranges were 3.4% (range, 1.1%-30%), 13.3% (range, 1.4%-42.3%), and 11.8% (range, 7.6%-52.3%) for nonprogressors, intermediate progressors, and rapid progressors, respectively (P = 0.11). In conclusion, hepatocyte cell cycle entry may be important in the pathogenesis of posttransplant HCV hepatitis. Early assessment of hepatocyte Mcm-2 expression could help identify patients at high risk for progressive fibrosis before it occurs.

Relation between hepatocyte G1 arrest, impaired hepatic regeneration, and fibrosis in chronic hepatitis C virus infection.

BACKGROUNDS & AIMS: An increased risk of hepatitis C virus (HCV)-related cirrhosis is associated with hepatic steatosis, older age, and high alcohol consumption, which could be explained by synergistic effects on cell proliferation. We aimed to investigate hepatocyte cell cycle state and phase distribution in chronic HCV infection. METHODS: Liver biopsy specimens diagnostic for chronic HCV (70), liver regeneration following transplant-related ischemic-reperfusion injury (15), and "normal" liver adjacent to colorectal cancer metastasis (10) were studied. Immunohistochemistry was used to detect cell cycle phase markers cyclin D1 (maximal in G 1 ), cyclin A (S), cyclin B1 (cytoplasmic during G 2 ) and phosphorylated histone 3 protein (mitosis), mini-chromosome maintenance protein 2 (Mcm-2; present throughout the cell cycle), and cyclin-dependent kinase inhibitor p21, which inhibits G 1 /S progression. RESULTS: Hepatocyte Mcm-2 expression was elevated in chronic HCV and liver regeneration (13% vs 26.4%) but negligible in "normal" liver. In proportion to Mcm-2, there was no difference in cyclin D1 between chronic HCV infection and liver regeneration (51.6% of Mcm-2-positive hepatocytes vs 52.6%). In contrast, there was a striking reduction in cyclin A (3% vs 16.3%), cyclin B1 (.4% vs 2.3%), and phosphorylated histone 3 protein (0% vs 3.8%) in chronic HCV infection compared with liver regeneration. In chronic HCV infection, Mcm-2 and p21 expression were associated with fibrosis stage and positive serum HCV RNA. CONCLUSIONS: The data are consistent with hepatocyte G 1 arrest in chronic HCV infection. This could impair hepatocellular function and limit hepatic regeneration.

Residual activity of mutant androgen receptors explains wolffian duct development in the complete androgen insensitivity syndrome.

Development of the Wolffian ducts (WD) into epididymides and vasa deferentia is dependent on testosterone. Patients with the complete androgen insensitivity syndrome (CAIS) are therefore not expected to develop these structures. However, WD derivatives have been described in cases of CAIS. It is thought that these may be remnants. This study assesses the degree of WD development in 33 patients with CAIS and investigates whether this development was androgen dependent. Epididymides and vasa deferentia were identified in 70% of patients with substitution mutations in the androgen receptor ligand-binding domain. They were more developed than epididymides and vasa deferentia from 16- to 20-wk-old male fetuses, suggesting that the WD had been stimulated to grow, rather than failed to regress. Receptors with substitutions in the ligand-binding domain were normally expressed and showed residual response to androgens in transactivation assays. Patients with premature stop codons or frameshift mutations, which prevented androgen receptor expression, or DNA-binding domain mutations that abolished transcriptional activity did not have epididymides or vasa deferentia. We hypothesize that mutant receptors with residual activity in vitro respond to high local testosterone concentrations in vivo, thereby stimulating WD development. The classification of androgen insensitivity in such patients should be considered severe rather than complete.

A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer.

An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.