The association of pre-treatment neutrophil to lymphocyte ratio with overall survival in patients with glioblastoma multiforme.

Neutrophil-lymphocyte ratio (NLR) is a marker of systemic inflammatory response and its elevation has recently been shown to be a poor prognostic factor in many malignancies including colon, prostate and bladder cancer. The primary aim of this study was to assess the prognostic impact of NLR in a clinically annotated cohort of patients with glioblastoma multiforme (GBM). We hypothesised that elevated NLR would be associated with worse prognosis. Between 2004 and 2009, 137 patients had surgery for GBM and were assessed for consideration of adjuvant therapy at our institution. Of these, 84 patients with an evaluable pre-corticosteroid full blood count result were identified and included in the final analysis. Median overall survival was 9.3 months (range 0.7-82.1). On univariate analysis, age >65 years, gender, ECOG performance status ≥2, frontal tumour, extent of surgical resection, completion of adjuvant chemoradiation protocol and NLR > 4 were significantly correlated with overall survival. Patients with NLR > 4, had a worse median overall survival at 7.5 months versus 11.2 months in patients with NLR ≤ 4 (hazard ratio 1.6, 95 % CI 1.00-2.52, p = 0.048). On multivariate analysis NLR > 4 remained an independent prognostic indicator for poor outcome. These data are an important reminder of the potential relevance of host immunity in GBM. In our cohort, NLR > 4 conferred a worse prognosis independent of other well established prognostic factors. If validated in other cohorts NLR may prove to be a useful addition in predicting prognosis in GBM patients. The demonstration that host immunity plays a role in GBM biology suggests that investigation of emerging therapies which modulate host immune response are warranted in this disease.

The role of biopsy in the diagnosis of infections of the central nervous system.

CNS infections require prompt appropriate therapy, but do not usually require tissue biopsy for diagnosis. We performed a 5 year audit of CNS infections which required brain or spinal biopsy to determine or confirm a diagnosis of CNS infection. Sixteen cases were identified in which clinical, radiological or additional investigations including culture, serology or PCR for the suspected specific infective agents were not diagnostic. 6 (37.5%) were bacterial abscesses presenting as space-occupying intracerebral lesions with a differential diagnosis of neoplasm. There were 3 (18.7%) cases of toxoplasmosis and 2 (12.5%) cases of aspergillosis. There was one case (6.2%) of herpes simplex encephalitis, one cysticercosis and one progressive multifocal leucoencephalopathy, all biopsied as possible neoplasms. There were 2 (12.5%) cases of spinal tuberculosis, one multifocal, mimicking neurofibromatosis. This review highlights the usefulness of targeted biopsy in the rapid diagnosis of CNS infections. It also emphasizes the lack of specificity of 'negative' culture and serology in certain cases, especially in the setting of immune-compromise.

Vaginal cytology following primary hysterectomy for cervical cancer: is it useful?

BACKGROUND: Vaginal intraepithelial neoplasia (VAIN) is usually detected in patients with synchronous or antecedent cervical or vulval intraepithelial or invasive cancer. VAIN has the potential to progress to malignancy. AIMS: To determine the incidence and severity and analyse the management of vaginal dysplasia in patients undergoing primary hysterectomy for cervical cancer. METHODS: A retrospective study (1984-1998) identified 210 primary invasive cervical cancers. One-hundred and twenty-three patients had a primary hysterectomy. RESULTS: In follow-up six patients were found to have dyskaryosis in a second vaginal smear. Biopsies in the six patients with colposcopic lesions showed VAIN II (n=2), VAIN III (n=1),VAIN III / possible early invasion (n = 1) and invasive carcinoma (n=2). One patient with recurrent squamous cancer received salvage radiotherapy and one with recurrent adenocarcinoma received high dose progestogens and topical 5-fluorouracil. CONCLUSION: All patients are disease-free at follow-up.

Atoh1 null mice show directed afferent fiber growth to undifferentiated ear sensory epithelia followed by incomplete fiber retention.

Inner ear hair cells have been suggested as attractors for growing afferent fibers, possibly through the release of the neurotrophin brain-derived neurotrophic factor (BDNF). Atoh1 null mice never fully differentiate hair cells and supporting cells and, therefore, may show aberrations in the growth and/or retention of their innervation. We investigated the distribution of cells positive for Atoh1- or Bdnf-mediated beta-galactosidase expression in Atoh1 null and Atoh1 heterozygotic mice and correlated the distribution of these cells with their innervation. Embryonic day (E) 18.5 Atoh1 null and heterozygotic littermates show Atoh1- and BDNF-beta-galactosidase-positive cells in comparable distributions in the canal cristae and the cochlea apex. Atoh1-beta-galactosidase-positive but only occasional Bdnf-beta-galactosidase-positive cells are found in the utricle, saccule, and cochlea base of Atoh1 null mutant mice. Absence of Bdnf-beta-galactosidase expression in the utricle and saccule of Atoh1 null mice is first noted at E12.5, a time when Atoh1-beta-galactosidase expression is also first detected in these epithelia. These data suggest that expression of Bdnf is dependent on ATOH1 protein in some but does not require ATOH1 protein in other inner ear cells. Overall, the undifferentiated Atoh1- and Bdnf-beta-galactosidase-positive cells show a distribution reminiscent of that in the six sensory epithelia in control mice, suggesting that ear patterning processes can form discrete patches of Atoh1 and Bdnf expression in the absence of ATOH1 protein. The almost normal growth of afferent and efferent fibers in younger embryos suggests that neither fully differentiated hair cells nor BDNF are necessary for the initial targeted growth of fibers. E18.5 Atoh1 null mice have many afferent fibers to the apex of the cochlea, the anterior and the posterior crista, all areas with numerous Bdnf-beta-galactosidase-positive cells. Few fibers remain to the saccule, utricle, and the base of the cochlea, all areas with few or no Bdnf-beta-galactosidase-positive cells. Thus, retention of fibers is possible with BDNF, even in the absence of differentiated hair cells.

Evaluation of liquid-based cytology in cervical screening of high-risk populations: a split study of colposcopy and genito-urinary medicine populations.

A split study evaluated the ThinPrep(R) PapTesttrade mark (TP; Cytyc Corp., Boxborough, MA) compared with current methodologies of cervical cytology in two high-risk cohorts. One thousand, three hundred cases from a colposcopy clinic and a genito-urinary medicine outpatient clinic were examined. The TP reported increased detection of all grades of dyskaryosis (mild, moderate and severe; + 4.5%) and a decrease in borderline and unsuitable cases (- 4.9%). Four cases of high-grade dyskaryosis (moderate or severe) were detected only using the TP, while an additional four cases classified as high-grade dyskaryosis with the TP were reported as borderline by our conventional methods. The split-study finding of increased sensitivity with the TP provides for improved clinical management of patients in our high-risk cohorts.

Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.

The mouse small intestinal epithelium consists of four principal cell types deriving from one multipotent stem cell: enterocytes, goblet, enteroendocrine, and Paneth cells. Previous studies showed that Math1, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the gut. We find that loss of Math1 leads to depletion of goblet, enteroendocrine, and Paneth cells without affecting enterocytes. Colocalization of Math1 with Ki-67 in some proliferating cells suggests that secretory cells (goblet, enteroendocrine, and Paneth cells) arise from a common progenitor that expresses Math1, whereas absorptive cells (enterocytes) arise from a progenitor that is Math1-independent. The continuous rapid renewal of these cells makes the intestinal epithelium a model system for the study of stem cell regeneration and lineage commitment.

Proprioceptor pathway development is dependent on Math1.

The proprioceptive system provides continuous positional information on the limbs and body to the thalamus, cortex, pontine nucleus, and cerebellum. We showed previously that the basic helix-loop-helix transcription factor Math1 is essential for the development of certain components of the proprioceptive pathway, including inner-ear hair cells, cerebellar granule neurons, and the pontine nuclei. Here, we demonstrate that Math1 null embryos lack the D1 interneurons and that these interneurons give rise to a subset of proprioceptor interneurons and the spinocerebellar and cuneocerebellar tracts. We also identify three downstream genes of Math1 (Lh2A, Lh2B, and Barhl1) and establish that Math1 governs the development of multiple components of the proprioceptive pathway.

Expression profile of human herpesvirus 8 (HHV-8) in pyothorax associated lymphoma and in effusion lymphoma.

AIMS: Pyothorax associated lymphoma (PAL) occurs in a clinical setting of longstanding pyothorax or chronic inflammation of the pleura. Like primary effusion lymphoma, it has an association with Epstein-Barr virus (EBV), and is confined to the pleural cavity, but has differing morphological and phenotypic features. Human herpesvirus 8 (HHV-8) has been consistently reported in primary effusion lymphoma. This study examines the immunophenotype of two European cases of PAL, investigates the presence of HHV-8 and its expression profile, and assesses whether PAL is similar to other effusion lymphomas. METHODS: Material was obtained from two European cases of PAL. Immunocytochemical analysis was performed using antibodies against CD45, CD20, CD79a, CD45RAA, CD3, CD43, CD45RO (UCHL1), CD30, BCL-2, CD68, epithelial membrane antigen (EMA), BCL-6, p53, Ki-67, kappa light chain, lambda light chain, and the EBV antigens latent membrane protein 1 (LMP-1) and EBV encoded nuclear antigen 2 (EBNA-2). The cases were examined for HHV-8 by means of polymerase chain reaction in situ hybridisation (PCR-ISH), solution phase PCR, in situ hybridisation (ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26) and viral (v) cyclin encoding regions. The expression profile of HHV-8 in PAL and in BC-1 and BC-3 cells was assessed by RNA TaqMan PCR to the HHV-8 genes encoding v-cyclin, v-IL-6, and G protein coupled receptor (GPCR). RESULTS: Both cases expressed CD24, CD20, CD79a, BCL-2, light chain restriction, and high Ki-67 staining. EBV was identified by EBER-ISH in one case. HHV-8 was not identified by solution phase PCR, but was detected by PCR-ISH (sensitivity of 1 viral genome copy/cell) in 35% of the cells and by TaqMan PCR, which showed 50-100 HHV-8 copies/2,000 cell genome equivalents (sensitivity of 1 viral genome in 10(6) contaminating sequences). HHV-8 v-IL-6, v-cyclin, and GPCR encoded transcripts were identified using RNA TaqMan PCR. v-IL-6 was high in PAL and in BC-1 and BC-3 cells. CONCLUSION: The presence of HHV-8 in one of two patients with PAL raises interesting questions in relation to the pathobiology of the condition. Clearly, the results indicate that HHV-8 is not an obligate pathogen, necessary for the effusion phenotype, but might contribute to it by its secretion of specific cytokines.

Evaluation of an SOS-Chromotest-based approach for the isolation and detection of sediment-associated genotoxins.

A series of experiments was conducted to evaluate an approach advanced by the St. Lawrence Centre (SLC) of Environment Canada for assessing the genotoxic potential of sediments. The SLC method entails the extraction, isolation and solvent exchange of the organic constituents in sediment, and the testing of these solubilized extracts with the SOS Chromotest (Escherichia coli PQ37). A total of five sediments, three variously contaminated by organic compounds and two reference materials certified for persistent organic chemicals, were Soxhlet-extracted. Each of the five extracts was then split, with a portion remaining in crude form and another portion fractionated into two molecular-weight classes of organic contaminants, thus yielding a total of 15 extract samples. The ability of the SOS Chromotest to detect genotoxins in the various organic extracts was evaluated and compared with that of the Ames Fluctuation Assay (Salmonella typhimurium, strain TA100). The intra-laboratory variance associated with the SOS Chromotest was also assessed. Procedural details are presented and results are discussed. The SOS Chromotest results were in good agreement with those of the Ames Fluctuation Assay, especially after metabolic activation. However, the E. coli PQ37 system was slightly more sensitive than the Salmonella assay for detecting genotoxins in the sediment extracts. The SOS Chromotest was also the most discriminating of the two assays, generating SOS-induction factors that were consistent with the organic contamination gradient reported in the sediment samples. The removal of macromolecules from the dichloromethane extracts by size-exclusion chromatography prior to testing enhanced the sensitivity of both test systems. The intra-laboratory variance of the SOS Chromotest ranged from 0.24% to 23.82%, depending on the extract sample. As applied in this study, the SOS Chromotest can serve as a sensitive test for screening the genotoxic potential of uncharacterized sediment extracts. A more sensitive assay would be appropriate, however, as a confirmation for definitive investigations, especially for the detection of direct-acting genotoxins.

Developmental evolutionary biology of the vertebrate ear: conserving mechanoelectric transduction and developmental pathways in diverging morphologies.

This brief overview shows that a start has been made to molecularly dissect vertebrate ear development and its evolutionary conservation to the development of the insect hearing organ. However, neither the patterning process of the ear nor the patterning process of insect sensory organs is sufficiently known at the moment to provide more than a first glimpse. Moreover, hardly anything is known about otocyst development of the cephalopod molluscs, another triploblast lineage that evolved complex 'ears'. We hope that the apparent conserved functional and cellular components present in the ciliated sensory neurons/hair cells will also be found in the genes required for vertebrate ear and insect sensory organ morphogenesis (Fig. 3). Likewise, we expect that homologous pre-patterning genes will soon be identified for the non-sensory cell development, which is more than a blocking of neuronal development through the Delta/Notch signaling system. Generation of the apparently unique ear could thus represent a multiplication of non-sensory cells by asymmetric and symmetric divisions as well as modification of existing patterning process by implementing novel developmental modules. In the final analysis, the vertebrate ear may come about by increasing the level of gene interactions in an already existing and highly conserved interactive cascade of bHLH genes. Since this was apparently achieved in all three lineages of triploblasts independently (Fig. 3), we now need to understand how much of the morphogenetic cascades are equally conserved across phyla to generate complex ears. The existing mutations in humans and mice may be able to point the direction of future research to understand the development of specific cell types and morphologies in the formation of complex arthropod, cephalopod, and vertebrate 'ears'.

Localisation of HHV-8 in AIDS related lymphadenopathy.

BACKGROUND: Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposi's sarcoma (KS), malignant lymphoma (Hodgkin's and non-Hodgkin's), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. AIM: To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposi's sarcoma development. METHODS: A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). RESULTS: Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. CONCLUSIONS: The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

Improved in situ detection method for telomeric tandem repeats in metaphase spreads and interphase nuclei.

Peptide nucleic acid technology (PNA) has become an extremely useful tool and promises to impact on molecular biology and diagnostics. These synthetic DNA analogues pair with DNA and RNA molecules according to Watson and Crick base pairing rules. This paper describes a sensitive and quick fluorescent in situ hybridisation (ISH) technique to determine DNA telomere repeat sequences (TTA GGG)n using epifluorescence microscopy. Telomeres are special, repeated structures at the end of each eukaryotic chromosome and serve as protective caps to prevent DNA rearrangements and fusion of chromosomes. A model system has been developed, using stimulated peripheral blood lymphocytes, which facilitates simultaneous detection of telomeres in metaphase as well as in interphase nuclei. A fluorescein isothiocyanate labelled PNA probe (18 mer) directed against complementary telomeric sequences at the end of each chromosome is used. In addition, a simple, easy to perform PNA-ISH protocol is described that overcomes common hybridisation problems encountered using DNA and RNA oligoprobes. Furthermore, the usefulness of a chromogenic immunocytochemical detection system is shown for PNA-ISH.

Cellular localisation of HHV-8 in Castleman's disease: is there a link with lymph node vascularity?

AIMS: Human herpesvirus 8 (HHV-8) has been identified in multicentric Castleman's disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis. METHODS: Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as "low" or "high". RESULTS: Five multicentric Castleman's disease and two solitary Castleman's disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10-50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. CONCLUSION: The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.

atonal regulates neurite arborization but does not act as a proneural gene in the Drosophila brain.

Drosophila atonal (ato) is the proneural gene of the chordotonal organs (CHOs) in the peripheral nervous system (PNS) and the larval and adult photoreceptor organs. Here, we show that ato is expressed at multiple stages during the development of a lineage of central brain neurons that innervate the optic lobes and are required for eclosion. A novel fate mapping approach shows that ato is expressed in the embryonic precursors of these neurons and that its expression is reactivated in third instar larvae (L3). In contrast to its function in the PNS, ato does not act as a proneural gene in the embryonic brain. Instead, ato performs a novel function, regulating arborization during larval and pupal development by interacting with Notch.

Functional conservation of atonal and Math1 in the CNS and PNS.

To determine the extent to which atonal and its mouse homolog Math1 exhibit functional conservation, we inserted (beta)-galactosidase (lacZ) into the Math1 locus and analyzed its expression, evaluated consequences of loss of Math1 function, and expressed Math1 in atonal mutant flies. lacZ under the control of Math1 regulatory elements duplicated the previously known expression pattern of Math1 in the CNS (i.e., the neural tube, dorsal spinal cord, brainstem, and cerebellar external granule neurons) but also revealed new sites of expression: PNS mechanoreceptors (inner ear hair cells and Merkel cells) and articular chondrocytes. Expressing Math1 induced ectopic chordotonal organs (CHOs) in wild-type flies and partially rescued CHO loss in atonal mutant embryos. These data demonstrate that both the mouse and fly homologs encode lineage identity information and, more interestingly, that some of the cells dependent on this information serve similar mechanoreceptor functions.

The kinesin light chain gene: its mapping and exclusion in mouse and human forms of inherited motor neuron degeneration.

The underlying genetic cause is known for only 10-20% of familial motor neuron disease (MND). Thus the genes involved in the aetiology of 80-90% of familial MND remain to be determined, and animal models are powerful tools for undertaking this task. We have mapped a heritable form of motor neuron degeneration in the mouse to a region that has homology to human chromosome 14q32.1-qter. This region contains the kinesin light chain gene (KLC1), which is a candidate for involvement in motor neuron degeneration because of its function in the motor-protein kinesin, and its neuronal expression. To investigate the role of KLC1 in a mouse motor neuron degeneration mutant that we are studying, we have identified mouse Klc1 gene sequences and mapped them with respect to our mutant locus. We have also investigated KLC1 in human patients with familial MND. Based on recombination and the absence of mutations in the coding region of KLC1, this gene can be excluded as a candidate gene in our mouse mutation and, where we have investigated, it is normal in human familial MND.