Liquid-based cytology for primary cervical cancer screening: a multi-centre study.

The aim of this six-centre, split-sample study was to compare ThinPrep fluid-based cytology to the conventional Papanicolaou smear. Six cytopathology laboratories and 35 gynaecologists participated. 5428 patients met the inclusion criteria (age > 18 years old, intact cervix, informed consent). Each cervical sample was used first to prepare a conventional Pap smear, then the sampling device was rinsed into a PreservCyt vial, and a ThinPrep slide was made. Screening of slide pairs was blinded (n = 5428). All non-negative concordant cases (n = 101), all non-concordant cases (n = 206), and a 5% random sample of concordant negative cases (n = 272) underwent review by one independent pathologist then by the panel of 6 investigators. Initial (blinded) screening results for ThinPrep and conventional smears were correlated. Initial diagnoses were correlated with consensus cytological diagnoses. Differences in disease detection were evaluated using McNemar's test. On initial screening, 29% more ASCUS cases and 39% more low-grade squamous intraepithelial lesions (LSIL) and more severe lesions (LSIL+) were detected on the ThinPrep slides than on the conventional smears (P = 0.001), including 50% more LSIL and 18% more high-grade SIL (HSIL). The ASCUS:SIL ratio was lower for the ThinPrep method (115:132 = 0.87:1) than for the conventional smear method (89:94 = 0.95:1). The same trend was observed for the ASCUS/AGUS:LSIL ratio. Independent and consensus review confirmed 145 LSIL+ diagnoses; of these, 18% more had been detected initially on the ThinPrep slides than on the conventional smears (P = 0.041). The ThinPrep Pap Test is more accurate than the conventional Pap test and has the potential to optimize the effectiveness of primary cervical cancer screening.

[Liquid phase cytology in the primary screening for cervical cancer: a multicenter study]. / Cytologie en phase liquide dans le cadre du dépistage primaire du cancer du col utérin: une étude multicentrique.

The aim of this six-centre, split-sample study was to compare ThinPrep fluid-based cytology to the conventional Papanicolaou smear. Six Cytopathology laboratories and 35 Gynaecologists participated. 5428 patients met the inclusion criteria. Each cervical sample was used first to prepare a conventional Pap smear, then the sampling device was rinsed into a PreservCyt vial, and a ThinPrep slide was made. Screening of slide pairs was blinded. On initial screening, 29% more ASCUS and 39% more low-grade squamous intraepithelial lesions (LSIL) and more severe lesions (LSIL+) were detected on the ThinPrep slides than on the conventional smears (p = 0.001). Independent and consensus review confirmed 145 LSIL + diagnoses; of these, 18% more had been detected initially on the ThinPrep slides than on the conventional smears (p = 0.041). The ThinPrep Pap Test is more accurate than the conventional Pap Test and has the potential to optimize the effectiveness of primary cervical cancer screening.

Expression of the mRNA for the beta 2 subunit of the voltage-dependent sodium channel in rat CNS.

Expression of the voltage-dependent sodium channel has been analysed in adult rat central nervous system by Northern blotting and in situ hybridization. Northern blots showed that all the territories studied express beta 2 transcripts, albeit with widely varying levels (with cerebellum >> hippocampus > brain > brainstem > spinal cord). In situ hybridization confirmed that in these structures, all the neuronal cell bodies contain beta 2 mRNA; expression was particularly high in the granule cells of the cerebellum, in both pyramidal cell layer and dentate gyrus in the hippocampus, and in spinal cord motor neurons. Northern blots also showed that RNA extracted from optic nerve and cultured cortical astrocytes contained beta 2 mRNA, while it was totally absent from sciatic nerve. In situ hybridization evidenced the presence of a numerous population of beta 2-positive cells in cerebellum white matter, spinal cord white matter, and in corpus callosum, where frontal sections showed labelled cells arranged in the chain-like or row pattern typical of interfascicular oligodendrocytes. Combination of antiglial fibrillary acid protein (GFAP) immunofluorescent histochemistry with detection of beta 2 mRNA evidenced that expression of the transcripts was indeed restricted to GFAP-negative cells in white matter.

HPV typing by in situ hybridization on cervical cytologic smears with ASCUS.

OBJECTIVE: To assess the prognostic significance of atypical squamous cells of undetermined significance (ASCUS) using an in situ hybridization (ISH) method for destined cervical cytologic smears and a cocktail of biotinylated DNA probes for human papillomavirus (HPV) 6, 11, 16, 18, 31 and 33. STUDY DESIGN: Two HPV DNA probe mixtures were applied to the same smear for the simultaneous detection of high-risk HPV types 16, 18, 31 and 33 and low-risk HPV 6 and 11. ISH was carried out on 192 smears. Among them, 59 showed koilocytosis, 91 ASCUS and 42 normal features. RESULTS: Low-risk HPV types were rarely found and associated mainly with koilocytosis (17%). High rates of potentially oncogenic HPV were detected in ASCUS (41%) and condyloma (73%). In addition, similar levels of positivity were found to be associated with ASCUS when using two probe mixtures specific to high-risk HPV: one included HPV genotypes 16 and 18 and the other, genotypes 31 and 33. CONCLUSION: HPV DNA typing by ISH on cervical cytologic smears might improve the identification of women at high risk of developing precancerous and cancerous cervical lesions.

Diagnosis of ventilator-associated pneumonia: an evaluation of direct examination and presence of intracellular organisms.

BACKGROUND: Ventilator-associated pneumonia (VAP) requires early diagnosis and adequate antibiotic therapy. The aim of this prospective postmortem study was to assess the accuracy of direct examination and quantification of intracellular organisms (ICO) for the diagnosis of VAP. METHODS: Total and differential cell counts were performed on fluids recovered using nonbronchoscopic sampling techniques (blind bronchial sampling [BBS], mini-bronchoalveolar lavage [mini-BAL]) and from bronchoalveolar lavage (BAL) performed during fiberscopy. These 3 sampling techniques were done within 15 min of death without discontinuing mechanical ventilation. Quantification of ICO was performed on each sample recovered from the various sampling procedures. Gram reaction and morphology of bacteria were evaluated on Gram smears. RESULTS: The results of each technique were compared with histology and culture of lung tissue specimens obtained by surgical pneumonectomies in 28 patients who died after at least 72 h of mechanical ventilation. Histology was positive in 13 patients and negative in 15 patients. When only VAP with positive lung culture was considered (histologic signs of bronchopneumonia plus positive lung tissue culture), the sensitivity of Gram staining on BAL, mini-BAL, and BBS was 56%, 44%, and 56%, respectively. If all samples were considered, the sensitivity and the specificity of the determination of the percentage of ICO were low (less than 70%) whatever the sampling technique. CONCLUSIONS: For initial therapeutic guidance, direct examination and presence of ICO do not contribute for establishing the diagnosis of VAP, essentially because of a lack of sensitivity. However, when positive, Gram staining can obviously guide initial antibiotherapy.

Astrocyte-regulated synaptogenesis: an in vitro ultrastructural study.

Striatal neurons from E15 rat embryos were dissociated, plated at low cell density on polyornithine or on astrocyte monolayers derived from the striatum (homotopic) or mesencephalon (heterotopic), and cultured in a chemically defined medium. Dendrites developing in homotopic co-cultures could reach a state of maturation allowing the establishment of synapses with axons from mesencephalic explants. This culture system thus partially reproduces the in vivo conditions in which striatal neurons developing in an homotopic glial environment can serve as synaptic targets for afferent mesencephalic axons.

Astrocyte-regulated GABA-ergic striatal neurons development: an in vitro ultrastructural study.

Striatal neurons from E15 rat embryos were dissociated, plated at low cell density on polyornithine or on astrocyte monolayers derived from the striatum (homotopic) or mesencephalon (heterotopic), and cultured in a chemically defined medium. After 2 to 10 days neurons could be divided in 3 classes according to their cell body diameter: small, medium or large. The percentage of small neurons which was very high 60% for GABAergic neurons on polyornithine after 2 days in vitro was reduced to 35% on mesencephalic astrocytes and to less than 20% on striatal astrocytes. The decrease in the number of small cells was paralleled by an increase in the number of multipolar medium size cells whereas the percentages of bipolar medium size and large neurons remained constant (55 and 4% respectively). All results obtained with the general neuronal population were replicated with the GABAergic sub-population which accounted for more than 50% of total neuronal population. These experiments confirm the beneficial influence of homotopic astrocytes on neuronal differentiation and on dendrite growth.

[Four years of screening cervical smears of cervical lesions]. / Quatre années de dépistage sur frottis cervical des lésions du col utérin.

Twelve thousand two hundred and eighty nine Pap smears were collected from public hospitals and from private practices during a four year period (January 1987 to December 1990). 4.2% of Pap smears exhibited condylomatous or dysplastic lesions. 94.5% of such lesions were encountered in Pap smears taken from the transformation zone and which contained endocervical cells. Therefore, these smears represent the only adequate sample for cervical cancer screening. In our study, a close concertation between biologists and clinicians results in an improvement of the smear quality. The percentage of those containing endocervical cells increased from 49% in 1987 to 72% in 1990. Then, more cervical lesions were encountered on smears of patients from a low socio-economic level. New techniques such as detection of human papillomavirus (HPV) DNA on routine Pap smears by in situ hybridization would allow to improve the cytological diagnosis of HPV infections, mainly for non specific cytological alterations (11% in our series for 1990) and for cytological aspects of dysplasia only. These results point out how a cervical cancer screening can be better carried out.

In vitro regulation of neuronal morphogenesis and polarity by astrocyte-derived factors.

Mesencephalic neurons were cultured from 2 to 5 days in mesencephalic (CM Gmes) or striatal (CM Gstr) astrocyte conditioned media or in the soluble (S100) and insoluble (P100) fractions prepared from these media by ultracentrifugation. CM Gmes as well as all soluble fractions induced dendritic and axonal elongation, whereas CM Gstr and the insoluble fractions promoted axonal growth only. The study of the shape of the neuronal cell bodies and the measurement of their adhesion to the substratum revealed that axons elongated under low adhesion conditions, but that dendrite growth was highly dependent upon adhesion and spreading of the neuronal soma. This different dependency of axonal and dendritic elongation upon spreading is explained by a model in which we consider the respective viscosities of axons and dendrites. From these observations and speculations we propose that axons and dendrites have different modes of elongation and that the primary effect of the astrocyte-derived factors capable of regulating neuronal polarity is to modify the adhesion of the neurons to their culture substratum.

Cyclic AMP reduces adhesion of isolated neuronal growth cones from developing rat forebrain to an astrocytic cell line from embryonic mouse striatum.

We have recently shown that isolated neuronal growth cones from developing rat forebrain possess an appreciable activity of adenylate cyclase, producing cyclic adenosine monophosphate, which can be stimulated by various neurotransmitter receptor agonists and by forskolin [Lockerbie R. O., Hervé D., Blanc G., Tassin J. P. and Glowinski J. (1988) Devl Brain Res. 38, 19-25]. In the present study, we have investigated the effect of cyclic adenosine monophosphate in an in vitro adhesion assay established between [3H]GABA-labelled isolated growth cones and a Simian virus-40 transformed astrocytic cell line from embryonic mouse striatum. Adhesion of the isolated growth cones onto the astrocytic clone increased steadily up to about 45 min before it began to level off at ca 16-18% of total [3H]GABA-labelled isolated growth cones added. Adhesion of the isolated growth cones onto the astrocytic clone was much superior to that seen on polyornithine and, in particular, on non-treated tissue culture wells. Adhesion "at plateau" was independent of both temperature and extracellular Ca2+ and was markedly reduced (ca 50%) by trypsin pre-treatment of the isolated growth cones. Pre-treatment of the isolated growth cones with either forskolin or lipophilic analogues of cyclic adenosine monophosphate attenuated adhesion in a time- and concentration-dependent manner. Approximately 30% reduction in adhesion to the astrocytic clone "at plateau" was observed after a 15 min pre-treatment of the isolated growth cones with forskolin at 10(-4) M or cyclic adenosine monophosphate analogues at 10(-3) M. A cyclic guanosine monophosphate analogue was without effect on adhesion of isolated growth cones. Scanning electron microscope analysis showed that isolated growth cones pre-treated with either cyclic adenosine monophosphate analogues or forskolin had a simpler morphology when attached to the astrocytic clone than isolated growth cones under control conditions. Pre-treatment of the isolated growth cones with low concentrations of cyclic adenosine monophosphate analogues increased protein kinase activity, measured using an exogenous histone phosphate acceptor, to a level which could not be further stimulated by cyclic adenosine monophosphate. Pre-treatment with a cyclic guanosine monophosphate analogue produced the same effect but only at much higher concentrations than those required for cyclic adenosine monophosphate analogues.

Region-specific neuro-astroglial interactions: ultrastructural study of the in vitro expression of neuronal polarity.

Mesencephalic neurons were cultured for 2 days on mesencephalic or striatal astrocyte monolayers. The morphology of these neurons was studied in electron microscopy. The number of dendritic profiles was higher on mesencephalic astrocytes (homotopic neuro-astroglial co-cultures) than on striatal astrocytes (heterotopic co-cultures). This increase in the number of dendrites correlated with a more mature aspect of the neurons. Striatal neurons were also cultured on the astrocytic monolayers. The state of maturation of these neurons was more advanced, and the number of their dendrites was higher on striatal than on mesencephalic astrocytes. These results confirm and extend the fact that neuronal maturation and dendritic growth can be regulated through region-specific neuro-astroglial interactions (Denis-Donini et al., 1984; Chamak et al., 1987).

[Role of neuro-astroglial interactions in the in vitro maturation of embryonic mesencephalic neurons: electron microscopic and radioautographic study]. / Rôle des interactions neuro-astrogliales dans la maturation in vitro des neurones mesencéphaliques embryonnaires: étude en microscopie électronique et par radioautographie.

Embryonic mouse neurons from the mesencephalon were plated on astrocyte monolayers from the mesencephalon and the striatum. Using ultrastructural criteria, it is shown that neuronal maturation and dendritic outgrowth are stimulated when neurons and astrocytes derive from the same structure. This is true for the dopaminergic neurons visualized by autoradiography after 3H-DA uptake. It is also true for the non-DA neurons present in these cultures. This finding raises the possibility that astrocytes synthetize specific neuronotrophic agents which act primarily upon regionally co-located neuronal subpopulation.

Two simian virus 40 (SV40)-transformed cell lines from the mouse striatum and mesencephalon presenting astrocytic characters. III. A light and electron microscopic study.

In two preceding papers we described the cloning of two astrocytic cell lines by simian virus 40 (SV40) transformation of embryonic mouse mesencephalon (F7-Mes) and striatum (F12-Str). The characterization of these lines as belonging to the astrocytic lineage is based on pharmacological, immunocytochemical and physiological data. Here we present quantitative and qualitative data on the morphological aspects of these two astrocytic clones observed under light and electron microscopy. We show that the clones present ultrastructural characters reminiscent of the morphology of young astrocytes. On one hand, they are rather similar to primary astrocytes in culture; on the other, they differ both from a clonal fibroblastic cell line (BT2) and from embryonic mouse fibroblasts in primary culture. These astroblastic clones display 4 morphologically different cell populations which we called types I, II, III and IV. Types II and III are very similar and represent the most predominant cells; their morphologies strongly remind of that of astroblasts. Type I corresponds to glioblasts and does not account for more than 15-20% of the total population. Type IV, which is very similar to differentiated velamentous astrocytes, normally represent ca. 5% of the cells. However, when the transformed cells are treated with mitomycin or mitomycin + dibutyryl cyclic AMP (dbcAMP), the proportion of type IV cells increases very much (up to more than 50% of the cells) while types I, II and III become less numerous. Morphological analysis therefore confirms that the two cell lines derived from the SV40 transformation of 14-day-old embryonic mesencephalic and striatal cells belong to the astrocytic lineage. Moreover, it seems that they can differentiate in vitro in cell culture conditions either spontaneously or under the action of pharmacological treatments known to enhance normal astrocyte maturation.

A combined light and electron microscopic method for the visualization of the same in vitro neuron by radioautography and serial sections.

We report here on a technical improvement which makes it possible to study, at the ultrastructural level, a dopaminergic neuron which has been previously identified by light microscopy. Primary cultures of virtually pure mesencephalic neurons from mouse embryos were obtained. These cultures were kept for 6 days, then incubated with tritiated dopamine, fixed and embedded in Epon. The dopaminergic neurons were firstly visualized by radioautography directly through Epon blocks in toto by light microscopy. In a second step, ultrathin sections of the identified dopaminergic cells were prepared and the neurons observed at the electron microscopy level. The dopaminergic nature of these neurons was regularly checked by radioautographic control on some selected ultrathin sections.

A cytochemical and ultrastructural study of the "S.I.F." cells in cat sympathetic ganglia.

According to the hypothesis of Eccles and Libet, the small intensely fluorescent cells (S.I.F. cells) in the sympathetic ganglion would represent an essential element in the inhibition of the principal neuron. As a contribution to the study of this important problem, we have investigated serial sections in superior cervical (S.C.G.) and celiac (C.G.) ganglia of the cat, a species that has not been extensively studied up to now, both by fluorescence and electron microscopy. We have shown that the "S.I.F." cells are three times fewer in the cat S.C.G. than in the rat S.C.G. There are five times more "S.I.F." cells in the C.G. of the cat than in the S.C.G. of the same species. Moreover we have described two types of "S.I.F." cells. Type I is composed of cells characterized by highly polymorphous large dense-cored vesicles. These cells lack processes and are grouped in clusters centered on fenestrated capillaries. They could be endocrine function cells. Type II is formed of isolated cells which exibit long processes and establish synaptic junctions with the dendrites of the principal neurons. In this case, the dense-cored vesicles are very regular and much smaller. These cells could be equivalent to interneurons. Type I very strongly predominates in the S.C.G. and C.G. of the cat where it represents more than 90% of the "S.I.F." cell total observed by fluorescence microscopy. A priori such a quantitative and qualitative heterogeneity hardly consistent with Eccles and Libet's hypothesis based on the existence of dopaminergic interneurons only, allows the question to be raised as to the functional significance of the "S.I.F." cells in ganglion physiology. The notion of modulation of ganglionic transmission does not seem to be quiered by these new data but could be founded on different forms of action embodied in the broader conception of the neuromodulation phenomenon.