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1.  A case of systemic lupus erythematosus with sideroblastic anaemia terminating in erythroleukaemia. 
Annals of the Rheumatic Diseases  1981;40(4):422-426.
A case of systemic lupus erythematosus is described in which for 10 years the only significant findings were erythema multiforme and vasculitis. Gross hepatosplenomegaly with persistent pancytopenia developed, and bone marrow examination revealed the presence of sideroblasts. The patient's condition deteriorated, and subsequently she developed a severe bleeding tendency, terminating in erythroleukaemia.
PMCID: PMC1000742  PMID: 6942789
2.  An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530) 
Cell Death & Disease  2013;4(11):e915-.
The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.
PMCID: PMC3847320  PMID: 24201814
epithelial–mesenchymal transition; intermediate states; ovarian cancer
3.  Sleep Disturbance and Older Adults' Inflammatory Responses to Acute Stress 
Poor sleep diminishes mental and physical health. The objective of this study was to examine associations between sleep disturbance and interleukin-6 (IL-6) responses to acute mental stress in older adults.
Observational study of community-dwelling, healthy older adults.
Participants completed the study in a clinical research laboratory of a mid-sized university.
Generally healthy, community-dwelling men and women 50 years of age and older.
IL-6 and negative affect at rest and following a series of challenging cognitive tests; sleep quality; depressive symptoms; perceived stress; loneliness.
Participants categorized as poor sleepers based on Pittsburgh Sleep Quality Index scores had significantly larger IL-6 responses to the cognitive stressors compared to good sleepers. The association between poor sleep and heightened IL-6 response to acute stress was not explained by other psychosocial factors previously linked to immune dysregulation, including depressive symptoms, perceived stress, and loneliness.
Findings add to the growing evidence for poor sleep as an independent risk factor for poor mental and physical health. Older adults may be particularly vulnerable to effects of sleep disturbance due to significant age-related changes in both sleep and inflammatory regulation.
PMCID: PMC3588882  PMID: 22327621
sleep disturbance; stress; interleukin-6
4.  Regulation of Adaptive Immunity; The Role of Interleukin-10 
Since the discovery of interleukin-10 (IL-10) in the 1980s, a large body of work has led to its recognition as a pleiotropic immunomodulatory cytokine that affects both the innate and adaptive immune systems. IL-10 is produced by a wide range of cell types, but for the purposes of this review we shall focus on IL-10 secreted by CD4+ T cells. Here we describe the importance of IL-10 as a mediator of suppression used by both FoxP3+ and FoxP3− T regulatory cells. Moreover, we discuss the molecular events leading to the induction of IL-10 secretion in T helper cell subsets, where it acts as a pivotal negative feedback mechanism. Finally we discuss how a greater understanding of this principle has allowed for the design of more efficient, antigen-specific immunotherapy strategies to exploit this natural phenomenon clinically.
PMCID: PMC3668291  PMID: 23755052
allergy; autoimmunity; cytokines; immune regulation; immunotherapy; interleukin-10; regulatory T cells; T helper cells
5.  Association of TCF7L2 and ADIPOQ with Body Mass Index, Waist–Hip Ratio, and Systolic Blood Pressure in an Endogamous Ethnic Group of India 
Despite the increasing burden of type 2 diabetes (T2D) and its established association with anthropometric and physiological traits as a risk factor, genetic studies focusing on the association of T2D-related genes with quantitative traits like body mass index (BMI), waist–hip ratio (WHR), and systolic blood pressure (SBP) are only a few for western populations and rare for Indian populations. The present study tested the association of TCF7L2, HHEX, KCNJ11, and ADIPOQ with BMI, SBP, and WHR in men and women of the Aggarwal population of India and found a differential association of TCF7L2 (rs7903146, rs4506565, and rs12256372) and ADIPOQ (rs2241766 and rs1501299) genes with increasing BMI, SBP, and WHR between the two sexes. We conclude that TCF7L2 and ADIPOQ together might play an important role in explaining these traits and to understand the biological and genetic mechanisms underlying T2D, and the role of other T2D genes must also be evaluated with these continuous traits.
PMCID: PMC3710444  PMID: 22583123
6.  Chronic Low Back Pain, Sleep Disturbance, and Interleukin-6 
The Clinical journal of pain  2011;27(1):35-41.
Sleep disturbance is a common co-morbidity of chronic pain. Inflammatory processes are dysregulated in sleep disturbance and also contribute to pain sensitivity. Thus, inflammation may play an important role in bi-directional associations between pain and sleep. Little is known about concurrent relationships among chronic pain, sleep, and inflammation. The aim of our study was to examine associations among sleep disturbance and circulating levels of the inflammatory cytokine, interleukin-6 (IL-6), in individuals with and without chronic low back pain.
Gender and age-matched adults with chronic low back pain (CLBP; n = 25) or without chronic pain (controls; n = 25) completed measures of sleep quality in the past month and depressive symptoms in the past week, and provided a blood draw for IL-6. The next morning, participants reported their sleep quality the previous night and their current experience of morning pain.
Individuals with CLBP had more sleep disturbance than controls. Circulating IL-6 levels were similar for the two groups; however, in adults with CLBP, poorer sleep quality was associated with higher IL-6 levels, and both sleep and IL-6 related to pain reports. Unlike CLBP participants, controls showed normal, age-related increases in IL-6 levels, whereas sleep quality was unrelated to IL-6 levels. Depressive symptoms could not fully explain the observed associations.
Inflammatory processes may play a significant role in cycles of pain and sleep disturbance. Clinical interventions that improve sleep and reduce concomitant inflammatory dysregulation hold promise for chronic pain management.
PMCID: PMC3058637  PMID: 21188850
chronic back pain; sleep disturbance; inflammatory cytokines; interleukin-6
7.  Defaulters among lung cancer patients in a suburban district in a developing country 
Annals of Thoracic Medicine  2012;7(1):12-15.
This study was carried out to determine the prevalence, patient's characteristic and reasons for defaulting follow-up and treatment among patients with lung cancer.
Patients with histologically confirmed lung cancer were recruited. Patient's detailed demographic data, occupation, socioeconomic status, and educational level of both the patients and their children were recorded. Defaulters were classified as either intermittent or persistent defaulters. By using Chi-square test, defaulter status was compared with various demographic and disease characteristic factors. The reasons for default were determined.
Ninety five patients were recruited. Among them, 81.1% patients were males; 66.3% were Malays. The mean age (SD) was 60 ± 10.5 years. About 46.3% of the patients had Eastern Cooperation Oncology Group (ECOG) functional status 0/1 and 96.8% of the patients presented with advanced stage (Stage 3b or 4). Overall, 20 patients (21.1%) were defaulters (35.0% intermittent defaulters; 65.0% persistent defaulters). Among the intermittent defaulters, 8 patients defaulted once and one patient defaulted 3 times. Among the 20 defaulters, only 2 (10%) patients turned up for the second follow-up appointment after telephone reminder. Two main reasons for default were ‘too ill to come’ (38.5.5%) and logistic difficulties (23.1%). No correlation was found between patient education, children education, income, ECOG status, stage of the disease, race, and gender with the defaulter rate.
Defaulter rate among lung cancer patients was 21.1%. Children education level is the only significant factor associated with the defaulter rate.
PMCID: PMC3277034  PMID: 22347344
Children education level; defaulter; follow-up; lung cancer; phone reminder
8.  MCP-1 Antibody Treatment Enhances Damage and Impedes Repair of the Alveolar Epithelium in Influenza Pneumonitis 
Recent studies have demonstrated an essential role of alveolar macrophages during influenza virus infection. Enhanced mortalities were observed in macrophage-depleted mice and pigs after influenza virus infection, but the basis for the enhanced pathogenesis is unclear. This study revealed that blocking macrophage recruitment into the lungs in a mouse model of influenza pneumonitis resulted in enhanced alveolar epithelial damage and apoptosis, as evaluated by histopathology, immunohistochemistry, Western blot, RT-PCR, and TUNEL assays. Abrogation of macrophage recruitment was achieved by treatment with monoclonal antibody against monocyte chemoattractant protein-1 (MCP-1) after sub-lethal challenge with mouse-adapted human influenza A/Aichi/2/68 virus. Interestingly, elevated levels of hepatocyte growth factor (HGF), a mitogen for alveolar epithelium, were detected in bronchoalveolar lavage samples and in lung homogenates of control untreated and nonimmune immunoglobulin (Ig)G-treated mice after infection compared with anti–MCP-1–treated infected mice. The lungs of control animals also displayed strongly positive HGF staining in alveolar macrophages as well as alveolar epithelial cell hyperplasia. Co-culture of influenza virus–infected alveolar epithelial cells with freshly isolated alveolar macrophages induced HGF production and phagocytic activity of macrophages. Recombinant HGF added to mouse lung explants after influenza virus infection resulted in enhanced BrdU labeling of alveolar type II epithelial cells, indicating their proliferation, in contrast with anti-HGF treatment showing significantly reduced epithelial regeneration. Our data indicate that inhibition of macrophage recruitment augmented alveolar epithelial damage and apoptosis during influenza pneumonitis, and that HGF produced by macrophages in response to influenza participates in the resolution of alveolar epithelium.
PMCID: PMC2891499  PMID: 19617401
alveolar epithelial regeneration; apoptosis; hepatocyte growth factor; influenza pneumonitis; Monocyte chemoattractant protein-1
9.  Retroperitoneal ectopic pancreas: imaging findings 
The British Journal of Radiology  2009;82(984):e253-e255.
Ectopic pancreas is relatively uncommon and usually occurs in the stomach or duodenum. Retroperitoneal ectopic pancreas has not previously been documented. We report the case of a 48-year-old man with retroperitoneal ectopic pancreas that imitated bilateral adrenal tumours on ultrasound and MRI. Subsequent CT-guided biopsies confirmed an ectopic pancreas. The lesions remained stable during follow-up for 7 years. In retrospect, the similarity in signal intensities and enhancement pattern between the retroperitoneal masses and the pancreas may have been a clue to the diagnosis of this rare entity.
PMCID: PMC3473376  PMID: 19934067
10.  3D Segmentation and Quantification of a Masticatory Muscle from MR Data Using Patient-Specific Models and Matching Distributions 
A method is proposed for 3D segmentation and quantification of the masseter muscle from magnetic resonance (MR) images, which is often performed in pre-surgical planning and diagnosis. Because of a lack of suitable automatic techniques, a common practice is for clinicians to manually trace out all relevant regions from the image slices which is extremely time-consuming. The proposed method allows significant time savings. In the proposed method, a patient-specific masseter model is built from a test dataset after determining the dominant slices that represent the salient features of the 3D muscle shape from training datasets. Segmentation is carried out only on these slices in the test dataset, with shape-based interpolation then applied to build the patient-specific model, which serves as a coarse segmentation of the masseter. This is first refined by matching the intensity distribution within the masseter volume against the distribution estimated from the segmentations in the dominant slices, and further refined through boundary analysis where the homogeneity of the intensities of the boundary pixels is analyzed and outliers removed. It was observed that the left and right masseter muscles’ volumes in young adults (28.54 and 27.72cm3) are higher than those of older (ethnic group removed) adults (23.16 and 22.13cm3). Evaluation indicates good agreement between the segmentations and manual tracings, with average overlap indexes for the left and right masseters at 86.6% and 87.5% respectively.
PMCID: PMC3043717  PMID: 18516642
Segmentation; masseter; masticatory muscle; patient-specific; matching distribution
11.  Extraction Techniques for Polycyclic Aromatic Hydrocarbons in Soils 
This paper aims to provide a review of the analytical extraction techniques for polycyclic aromatic hydrocarbons (PAHs) in soils. The extraction technologies described here include Soxhlet extraction, ultrasonic and mechanical agitation, accelerated solvent extraction, supercritical and subcritical fluid extraction, microwave-assisted extraction, solid phase extraction and microextraction, thermal desorption and flash pyrolysis, as well as fluidised-bed extraction. The influencing factors in the extraction of PAHs from soil such as temperature, type of solvent, soil moisture, and other soil characteristics are also discussed. The paper concludes with a review of the models used to describe the kinetics of PAH desorption from soils during solvent extraction.
PMCID: PMC2853853  PMID: 20396670
12.  Specific epitopes of the structural and hypothetical proteins elicit variable humoral responses in SARS patients 
Journal of Clinical Pathology  2006;59(5):468-476.
Severe acute respiratory syndrome (SARS) is an infectious disease which was caused by a novel coronavirus (SARS‐CoV). SARS has caused an outbreak in the world during 2003 and 2004, with 8098 individuals being infected and a death toll of 774 in 28 regions around the world. Specific humoral responses to viral infection remain unclear.
To analyse the antigenicity of the SARS‐CoV genome and identify potential antigenic epitopes in the structural proteins.
Potential antigenic epitopes were identified in the structural proteins (nucleocapsid, membrane, spike, and small envelope proteins) and hypothetical proteins (SARS3a, 3b, 6, 7a, and 9b) that are specific for SARS‐CoV. A peptide chip platform was created and the profiles of antibodies to these epitopes were investigated in 59 different SARS patients' sera obtained 6–103 days after the onset of the illness. Serial sera from five additional patients were also studied.
Epitopes at the N‐terminus of the membrane protein and the C‐terminus of nucleocapsid protein elicited strong antibody responses. Epitopes on the spike protein were only moderately immunogenic but the effects were persistent. Antibodies were also detected for some putative proteins, noticeably the C‐termini of SARS3a and SARS6.
Important epitopes of the SARS‐CoV genome that may serve as potential markers for the viral infection are identified. These specific antigenic sites may also be important for vaccine development against this new fatal infectious disease.
PMCID: PMC1860290  PMID: 16461566
SARS; coronavirus; antibody; peptide chip
13.  Prolonged Mild-to-Moderate Hypothermia for Refractory Intracranial Hypertension 
Therapeutic hypothermia is an emerging therapy for brain injury and cerebral edema. Hypothermia is known to reduce death and neurologic morbidity in survivors of cardiac arrest from ventricular fibrillation. Traumatic brain injury (TBI) trials studies of short-term hypothermia (24 to 48hours) have had conflicting results. Recent evidence however suggests prolonged hypothermia (48 hours to 14 days) may be beneficial for TBI and select cases of nontraumatic brain injury especially when the duration of cerebral edema and intracranial hypertension is expected to last longer than 24 hours.
Case Report:
A 43-year-old female presented with a Fisher grade 4 aneurysmal (anterior communicating artery) subarachnoid hemorrhage. The patient was comatose upon transfer to our hospital, was intubated, and had immediate aneurysm coiling. The patient had a right external ventricular drain (EVD) placed for acute hydrocephalus and intracranial pressure (ICP) monitoring. The patient developed severe vasospasm of several intracranial vessels requiring angioplasty on two consecutive days, and hypertensive, hypervolemic, hemodilution therapy (HHH). On the ninth day, ICP went above 20mmHg and computed tomography (CT) showed global cerebral edema. For the next 17 days, the patient had refractory intracranial hypertension, requiring sedation, neuromuscular blockade, hyperosmolar therapy (3% infusion, and 23.4% saline boluses), thiopental coma with burst suppression, and hypothermia (31 to 34C). Hypothermia continued for a total of 14 days before ICP and edema on CT normalized.
We report the first case of prolonged therapeutic hypothermia over a total of 14days to control nontraumatic brain injury-related refractory intracranial pressure and global cerebral edema. More studies are needed comparing clinical outcomes and complication rates between short duration and prolonged hypothermia for brain injury.
PMCID: PMC3317328  PMID: 22518243
Intracranial hypertension; hypothermia; subarachnoid hemorrhage
14.  Glycoprotein D-Independent Spread of Pseudorabies Virus Infection in Cultured Peripheral Nervous System Neurons in a Compartmented System▿  
Journal of Virology  2007;81(19):10742-10757.
The molecular mechanisms underlying the directional neuron-to-epithelial cell transport of herpesvirus particles during infection are poorly understood. To study the role of the viral glycoprotein D (gD) in the directional spread of herpes simplex virus (HSV) and pseudorabies virus (PRV) infection, a culture system consisting of sympathetic neurons or epithelial cells in different compartments was employed. We discovered that PRV infection could spread efficiently from neurons to cells and back to neurons in the absence of gD, the viral ligand required for entry of extracellular particles. Unexpectedly, PRV infection can also spread transneuronally via axo-axonal contacts. We show that this form of interaxonal spread between neurons is gD independent and is not mediated by extracellular virions. We also found that unlike PRV gD, HSV-1 gD is required for neuron-to-cell spread of infection. Neither of the host cell gD receptors (HVEM and nectin-1) is required in target primary fibroblasts for neuron-to-cell spread of HSV-1 or PRV infection.
PMCID: PMC2045490  PMID: 17652377
15.  Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS) 
Journal of Clinical Pathology  2004;57(3):260-265.
Background: Severe acute respiratory syndrome (SARS) became a worldwide outbreak with a mortality of 9.2%. This new human emergent infectious disease is dominated by severe lower respiratory illness and is aetiologically linked to a new coronavirus (SARS-CoV).
Methods: Pulmonary pathology and clinical correlates were investigated in seven patients who died of SARS in whom there was a strong epidemiological link. Investigations include a review of clinical features, morphological assessment, histochemical and immunohistochemical stainings, ultrastructural study, and virological investigations in postmortem tissue.
Results: Positive viral culture for coronavirus was detected in most premortem nasopharyngeal aspirate specimens (five of six) and postmortem lung tissues (two of seven). Viral particles, consistent with coronavirus, could be detected in lung pneumocytes in most of the patients. These features suggested that pneumocytes are probably the primary target of infection. The pathological features were dominated by diffuse alveolar damage, with the presence of multinucleated pneumocytes. Fibrogranulation tissue proliferation in small airways and airspaces (bronchiolitis obliterans organising pneumonia-like lesions) in subpleural locations was also seen in some patients.
Conclusions: Viable SARS-CoV could be isolated from postmortem tissues. Postmortem examination allows tissue to be sampled for virological investigations and ultrastructural examination, and when coupled with the appropriate lung morphological changes, is valuable to confirm the diagnosis of SARS-CoV, particularly in clinically unapparent or suspicious but unconfirmed cases.
PMCID: PMC1770245  PMID: 14990596
pulmonary; severe acute respiratory syndrome; coronavirus
16.  Sputum cytology of patients with severe acute respiratory syndrome (SARS) 
Journal of Clinical Pathology  2004;57(3):256-259.
Background: Severe acute respiratory syndrome (SARS) is a newly described form of atypical pneumonia linked to a novel coronavirus.
Aims: To review the sputum cytology of 15 patients who fulfilled the World Health Organisation clinical criteria for SARS in an attempt to evaluate whether early diagnosis is feasible with routine sputum examination.
Methods: All sputum samples from patients with SARS from the four major hospitals in Hong Kong were reviewed; abnormalities were sought in the cellular component, including abnormal numbers and morphology of the component cells compared with those from age matched controls taken over the same period one year ago.
Results: Fifteen sputum samples from patients were compared with 25 control samples. In the patients with SARS, loose aggregates of macrophages were seen more frequently in the sputum. These macrophages frequently showed morphological changes, such as cytoplasmic foaminess, vacuole formation, and nuclear changes (including multinucleation and a ground glass appearance) when compared with the control samples.
Conclusions: The cytological features of SARS are non-specific, but the observation of any of the described features should prompt further investigations, especially in patients with suspicious clinical features.
PMCID: PMC1770235  PMID: 14990595
severe acute respiratory syndrome; coronavirus; sputum; cytology
17.  Role of Pseudorabies Virus Us3 Protein Kinase during Neuronal Infection 
Journal of Virology  2006;80(13):6387-6398.
The pseudorabies virus (PRV) Us3 gene is conserved among the alphaherpesviruses and encodes a serine/threonine protein kinase that is not required for growth in standard cell lines. In this report, we used a compartmented culture system to investigate the role of PRV Us3 in viral replication in neurons, in spread from neurons to PK15 cells, and in axon-mediated spread of infection. We also examined the role of Us3 in neuroinvasion and virulence in rodents. Us3 null mutants produce about 10-fold less infectious virus from neurons than wild-type virus and have no discernible phenotypes for axonal targeting of viral components in cultured peripheral nervous system neurons. After eye infection in rodents, Us3 null mutants were slightly attenuated for virulence, with a delayed onset of symptoms compared to the wild type or a Us3 null revertant. While initially delayed, the symptoms increased in severity until they approximated those of the wild-type virus. Us3 null mutants were neuroinvasive, spreading in both efferent and afferent circuits innervating eye tissues.
PMCID: PMC1488934  PMID: 16775327
18.  Identification of two contiguous minimally deleted regions on chromosome 1p36.31–p36.32 in oligodendroglial tumours 
British Journal of Cancer  2004;91(6):1105-1111.
Loss of the short arm of chromosome 1 is a hallmark of oligodendroglial tumours (OTs). Deletion mapping studies in OTs have revealed multiple commonly deleted regions on chromosome 1p, suggesting that there are more than one tumour suppressor gene. To map critical deletion regions on 1p, a series of 25 OTs were examined for loss of heterozygosity (LOH) on 19 polymorphic markers across the 1p arm using microsatellite analysis. Our study revealed that 60% of tumours had LOH of all informative markers on 1p and identified one tumour showing LOH at telomeric markers only. Since this deletion region lies in one of the critical deletion intervals defined previously, we then screened another series of 27 OTs specifically at 1p36.3 for LOH using nine polymorphic markers. A total of 12% (six out of 52) of tumours were found to carry interstitial deletions. The allelic status and the deletion breakpoints in these tumours with interstitial deletion were further verified by fluorescent in situ hybridisation. The small overlapping intervals facilitated the delineation of two contiguous minimally deleted regions of 0.76 Mb, defined by D1S468 and D1S2845, and of 0.41 Mb, bound by D1S2893 and D1S1608, on 1p36.31–36.32. Based on current reference human genome sequence these deletion regions have been sequenced almost to entirety and contain eight annotated genes. TP73, DFFB and SHREW1 are the only known genes located in these deletion regions, while the others are uncharacterised novel genes. In conclusion, our study has narrowed down the critical tumour suppressor loci on 1p36.3, in which two minimally deleted regions are mapped, and markedly reduced the number of candidate genes to be screened for their involvement in OT development.
PMCID: PMC2747719  PMID: 15475940
oligodendroglioma; oligoastrocytoma; tumour suppressor gene; loss of heterozygosity; chromosome 1p
19.  Neuron-to-Cell Spread of Pseudorabies Virus in a Compartmented Neuronal Culture System 
Journal of Virology  2005;79(17):10875-10889.
Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.
PMCID: PMC1193604  PMID: 16103140
20.  Efficient Axonal Localization of Alphaherpesvirus Structural Proteins in Cultured Sympathetic Neurons Requires Viral Glycoprotein E 
Journal of Virology  2005;79(14):8835-8846.
Pseudorabies virus (PRV) glycoprotein E (gE) is a type I viral membrane protein that facilitates the anterograde spread of viral infection from the peripheral nervous system to the brain. In animal models, a gE-null mutant infection spreads inefficiently from presynaptic neurons to postsynaptic neurons (anterograde spread of infection). However, the retrograde spread of infection from post- to presynaptic neurons remains unaffected. Here we show that gE is required for wild-type localization of viral structural proteins in axons of infected neurons. During a gE-null PRV infection, a subset of viral glycoproteins, capsids, and tegument proteins enter and localize to the axon inefficiently. This defect is most obvious in the distal axon and growth cones. However, axonal entry and localization of other viral membrane proteins and endogenous cellular proteins remains unaffected. Neurons infected with gE-null mutants produce wild-type levels of viral structural proteins and infectious virions in the cell body. Our results indicate that reduced axonal targeting of viral structural proteins is a compelling explanation for the lack of anterograde spread in neural circuits following infection by a gE-null mutant.
PMCID: PMC1168755  PMID: 15994777
21.  Heterogeneity of a Fluorescent Tegument Component in Single Pseudorabies Virus Virions and Enveloped Axonal Assemblies 
Journal of Virology  2005;79(7):3903-3919.
The molecular mechanisms responsible for long-distance, directional spread of alphaherpesvirus infections via axons of infected neurons are poorly understood. We describe the use of red and green fluorescent protein (GFP) fusions to capsid and tegument components, respectively, to visualize purified, single extracellular virions and axonal assemblies after pseudorabies virus (PRV) infection of cultured neurons. We observed heterogeneity in GFP fluorescence when GFP was fused to the tegument component VP22 in both single extracellular virions and discrete puncta in infected axons. This heterogeneity was observed in the presence or absence of a capsid structure detected by a fusion of monomeric red fluorescent protein to VP26. The similarity of the heterogeneous distribution of these fluorescent protein fusions in both purified virions and in axons suggested that tegument-capsid assembly and axonal targeting of viral components are linked. One possibility was that the assembly of extracellular and axonal particles containing the dually fluorescent fusion proteins occurred by the same process in the cell body. We tested this hypothesis by treating infected cultured neurons with brefeldin A, a potent inhibitor of herpesvirus maturation and secretion. Brefeldin A treatment disrupted the neuronal secretory pathway, affected fluorescent capsid and tegument transport in the cell body, and blocked subsequent entry into axons of capsid and tegument proteins. Electron microscopy demonstrated that in the absence of brefeldin A treatment, enveloped capsids entered axons, but in the presence of the inhibitor, unenveloped capsids accumulated in the cell body. These results support an assembly process in which PRV capsids acquire a membrane in the cell body prior to axonal entry and subsequent transport.
PMCID: PMC1061572  PMID: 15767393
22.  High-resolution genome-wide allelotype analysis identifies loss of chromosome 14q as a recurrent genetic alteration in astrocytic tumours 
British Journal of Cancer  2002;87(2):218-224.
Diffusely infiltrative astrocytic tumours are the most common neoplasms in the human brain. To localise putative tumour suppressor loci that are involved in low-grade astrocytomas, we performed high-resolution genome-wide allelotype analysis on 17 fibrillary astrocytomas. Non-random allelic losses were identified on chromosomal arms 10p (29%), 10q (29%), 14q (35%), 17p (53%), and 19q (29%), with their respective common regions of deletions delineated at 10p14-15.1, 10q25.1-qter, 14q212.2-qer, 17p11.2-pter and 19q12-13.4. These results suggest that alterations of these chromosomal regions play important roles in the development of astrocytoma. We also allelotyped 21 de novo glioblastoma multiforme with an aim to unveil genetic changes that are common to both types of astrocytic tumours. Non-random allelic losses were identified on 9p (67%), 10p (62%), 10q (76%), 13q (60%), 14q (50%), and 17p (65%). Allelic losses of 10p, 10q, 14q and 17p were common genetic alterations detectable in both fibrillary astrocytomas and glioblastoma multiforme. In addition, two common regions of deletions on chromosome 14 were mapped to 14q22.3-32.1 and 14q32.1-qter, suggesting the presence of two putative tumour suppressor genes. In conclusion, our comprehensive allelotype analysis has unveiled several critical tumour suppressor loci that are involved in the development of fibrillary astrocytomas and glioblastoma multiforme. Although these two types of brain tumours are believed to evolve from different genetic pathways, they do share some common genetic changes. Our results indicate that deletions of chromosome 14q is a recurrent genetic event in the development of astrocytoma and highlight the subchromosomal regions on this chromosome that are likely to contain putative tumour suppressor genes involved in the oncogenesis of astrocytic tumours.
British Journal of Cancer (2002) 87, 218–224. doi:10.1038/sj.bjc.6600430
© 2002 Cancer Research UK
PMCID: PMC2376105  PMID: 12107846
fibrillary astrocytoma; glioblastoma multiforme; allelotyping; loss of heterozygosity
23.  Antisense epidermal growth factor receptor RNA transfection in human glioblastoma cells down-regulates telomerase activity and telomere length 
British Journal of Cancer  2002;86(8):1328-1332.
Epidermal growth factor receptor is overexpressed and/or amplified in up to 50% of glioblastomas, suggesting an important role of this gene in glial tumorigenesis and progression. In the present study we demonstrated that epidermal growth factor receptor is involved in regulation of telomerase activity in glioblastoma. Antisense-epidermal growth factor receptor approach was used to inhibit epidermal growth factor receptor expression of glioblastoma U87MG cells. Telomerase activity in antisense-epidermal growth factor receptor cells decreased by up to 54 folds compared with control cells. Moreover, the telomere lengths of antisense-epidermal growth factor receptor cells were shortened. In addition, the tumorigenicity of antisense-epidermal growth factor receptor cells was significantly inhibited. Taken together, there were strong correlations between tumorigenicity and epidermal growth factor receptor expression levels, and between tumorigenicity and telomerase activity. These results provide evidence that epidermal growth factor receptor plays an important role in the regulation of telomerase activity of glioma cells. Our findings provide new insights into both the biological functions of epidermal growth factor receptor and the regulation of telomerase activity. The inhibition of telomerase activity triggered by antisense-epidermal growth factor receptor treatment may reflect yet another mechanism of antisense-epidermal growth factor receptor approach in tumour suppression.
British Journal of Cancer (2002) 86, 1328–1332. DOI: 10.1038/sj/bjc/6600244
© 2002 Cancer Research UK
PMCID: PMC2375350  PMID: 11953893
glioblastoma; epidermal growth factor receptor; antisense; telomerase
24.  Detection of human herpesviruses 6 and 7 genomic sequences in brain tumours. 
Journal of Clinical Pathology  1999;52(8):620-623.
BACKGROUND: Human herpesviruses 6 and 7 (HHV-6, HHV-7) are ubiquitous, with primary infection occurring early in life followed by persistence, which may involve neural tissue. While HHV-6 and HHV-7 are predominantly T lymphotropic, the extent of tissue tropism in persistent infection is not known. AIM: To investigate neuropersistence and the role of HHV-6 and HHV-7 in brain tumorigenesis. METHODS: Nested polymerase chain reaction was used to detect HHV-6 and HHV-7 genomic sequences in preparations of total DNA extracted from 98 formalin fixed, paraffin embedded primary brain tumours. HHV-6 detected was further characterized into variants A and B by restriction fragment length analysis. RESULTS: HHV-6 was detected in 8.2% of cases and HHV-7 in 14.3% (14/98). None of the positive samples contained both viruses. Among the eight HHV-6 positive tumours, three harboured variant A and five variant B. Four of the five ependymomas studied contained viral DNA. Otherwise, both HHV-6 and HHV-7 were present at similar low frequencies in most of the tumour types investigated. CONCLUSIONS: The findings do not support an aetiological role of HHV-6 and HHV-7 in primary brain tumour, but they suggest that HHV-6 and HHV-7 are neurotropic in vivo and that the central nervous system seems to be one of the reservoirs for persistent infection.
PMCID: PMC500955  PMID: 10645234
25.  Altered expression of the suppressors PML and p53 in glioblastoma cells with the antisense-EGF-receptor 
British Journal of Cancer  1999;81(6):994-1001.
Gene amplification and enhanced expression of the epidermal growth factor receptor (EGFR) represent the major molecular genetic alteration in glioblastomas and it may play an essential role in cell growth and in the carcinogenic process. On the other hand, the nuclear suppressor proteins PML and p53 are also known to play critical roles in cancer development and in suppressing cell growth. Here we report that, in glioblastoma cells with defective EGFR function, the expressions of both promyelocytic leukaemia (PML) and p53 were altered. Cells that were transfected with the antisense-cDNA of EGFR were found to have more cells in G1 and fewer cells in S phase. In addition, the transfected cells were found to be non-responsive to EGF-induced cell growth. Interestingly, the expression of the suppressors p53 and PML were found to be significantly increased by immunohistochemical assay in the antisense-EGFR cells. Moreover, the PML expression in many of the cells was converted from the nuclear dot pattern into fine-granulated staining pattern. In contrast, the expressions of other cell cycle regulated genes and proto-oncogene, including the cyclin-dependent kinase 4 (cdk4), retinoblastoma, p16INK4a and p21H-ras, were not altered. These data indicate that there are specific inductions of PML and p53 proteins which may account for the increase in G1 and growth arrest in antisense-EGFR treated cells. It also indicates that the EGF, p53 and PML transduction pathways were linked and they may constitute an integral part of an altered growth regulatory programme. The interactions and cross-talks of these critical molecules may be very important in regulating cell growth, differentiation and cellular response to treatment in glioblastomas. © 1999 Cancer Research Campaign
PMCID: PMC2362944  PMID: 10576656
PML; p53; antisense-EGFR; glioblastoma

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