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.

Early transient ultrastructural and quantitative changes in nucleolar vacuoles of sympathetic neurons in isolated superior cervical ganglion of the rat.

Nucleolar changes and their time-course following axotomy and decentralization were investigated using an ultrastructural and stereological analysis of sympathetic neurons in superior cervical ganglion. By comparison with ganglia fixed immediately after removal, incubation for 2 h, 3 h and 5 h in NCTC 109 medium at 37 degrees C was found to induce early changes in the nucleolar volume (first a 33% increase, followed by a 43% decrease and a subsequent recovery to control values after 5 h). Moreover, dense material (also called 'microspherules') appeared in nucleolar vacuoles as early as 2 h after the beginning of incubation and was present after 3 h, but disappeared after a 5 h incubation. The dense material may have been either fibrillar component involved in rDNA transcription, or granular component involved in the storage and/or transport of preribosomal particles. Such early, transient and quantitative changes were observed very earlier than in previous studies on the axon reaction.

[Multicentric bone chloroma disclosed by pleural cytology]. / Un chlorome multicentrique osseux révélé par la cytologie pleurale.

We report a case of granulocytic sarcoma of the bone with pleural involvement diagnosed upon cytologic analysis of the pleural fluid (centrifugation spots stained by May-Grunwald-Giemsa) and confirmed by more complex investigations, i.e., demonstration of granulomonocytic membrane antigens by immunohistochemical monoclonal antibody techniques on frozen sections of the tumor. This case draws attention to the value of cytologic studies in granulocytic sarcomas whose histologic features are suggestive of lymphoma.

Effects of isolation and high helium pressure on the nucleolus of sympathetic neurons in the rat superior cervical ganglion.

In prokaryotes, unicellular eukaryotes and cell-free systems, pressure is known to exert an inhibitory effect on protein synthesis and RNA metabolism, the mechanism(s) of which remain to be investigated in detail. The purpose of the present in vitro study was to compare ultrastructural and quantitative changes of the nucleolus, which is the site of ribosome biogenesis, in sympathetic neurons of rat superior cervical ganglia (SCG) maintained for 2, 3 and 5 h in NCTC 109 medium and subjected to pressure or not. In control SCG (left) the nucleolus greatly increased in volume (+ 33%) 2 h after excision, in comparison with SCG fixed immediately. This overall enlargement was found to reflect a marked increase in all nucleolar components (from 16 to 87%). After 5 h, volumes of nucleolus, fibrillar centers and vacuolar component returned to control values, whereas dense fibrillar and granular components remained affected. Such early and transient changes are regarded as reflecting basic metabolic changes associated with increased nucleolar RNA that should be of primary concern to experiments using SCG transplanted in culture media. Compression under helium up to 180 atmospheric pressure for 1 h of right SCG maintained for 2 h in culture medium, was shown to induce, on the contrary, a marked decrease in nucleolar volume (-39%) and in volumes of all nucleolar components (from -36 to -51%). When they were kept at constant high pressure for 1 and 3 h a progressive recovery of volumes of nucleoli and nucleolar components was observed. Consequently, compression was shown to exert opposite effects to those of isolation of SCG. Present data are interpreted as an inhibitory effect of pressure on ribosome biogenesis. Such observations on a vertebrate neuron might open a new field in the search for cellular mechanisms underlying the effects of pressure on living organisms and especially on the nervous system.

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.

Localization of transcription in nucleoli of rat sympathetic neurons. A quantitative ultrastructural autoradiography study.

A quantitative electron microscopy autoradiography method was used to locate the sites of initial rRNA transcription and to determine whether there was a variability of fibrillar centres size related to nucleolar transcription in nucleoli of sympathetic neurons of a rat killed during the dark span of its light-dark cycle. Indeed, in this model two types of fibrillar centres (nucleolar organizing regions) were observed: a single large type characteristic of this phase of the photoperiod and a small type composed by several small fibrillar centres of normal size. The present results clearly demonstrated that: there was no incorporation into the fibrillar centres themselves; the rRNA transcription sites were restricted to the ribonucleoprotein (RNP) fibrillar component and the products thereafter migrated into the RNP granular component. There was no variability of fibrillar centres size related to nucleolar transcription. The level of transcription was found to be identical around the two types of fibrillar centres. They might be considered as equivalent. The results provide evidence that at least in this model, the size of fibrillar centres and the transcriptional activity are not correlated. Other factors must therefore be responsible for the difference in size.

Changes in nucleoli and nucleolar fibrillar centres of chromaffin cells in rat adrenal medulla over a 24-hour period: an ultrastructural and stereological analysis.

A stereological and ultrastructural study was performed on the nucleoli of the adrenal medulla chromaffin cells of rats exposed to a standardized 12 h light/12 h dark cycle with free access to food and water. The animals were killed three at a time, every 4 h during the 24-h span and fixed by intracardiac perfusion. In these reticulated nucleoli, the stereological analysis over a 24-h period showed a variation dependent on the time of killing for the two parameters investigated, the mean nucleolar volume, Vnu, and the mean volume of the fibrillar centres, Vfc(nu). The minimal value occurred at 0300 h (dark span) and the maximal one at 0700 h (at the onset of the light span). Between these two extreme values, Vnu increased 1.8-fold and Vfc(nu) 5.3-fold. These data are compared with a previous description from our laboratory of circadian rhythm in nucleoli of sympathetic neurons of superior cervical ganglion in the same animals. Analogies and differences are pointed out, but apart from these considerations the present study provides a new example of temporal organization at the cellular level in the organelle involved in ribosomal RNA synthesis and ribosome assembly.

Ultrastructure and stereological analysis of nucleoli of rat nodose ganglion neuron during a 24-h period: a comparison with sympathetic neurons of rat superior cervical ganglion.

The nucleoli of rat nodose ganglion were investigated during a 24-h span (light span 07.00-19.00 h). The mean volume of nucleoli and that of their components, especially fibrillar centers considered to be the interphasic counterpart of nucleolus-organizing regions, were determined by stereological analysis. The quantitative data showed that (1) nucleoli volumes of rat nodose ganglion neurons did not oscillate diurnally but that (2) there were diurnal dimensional changes in the volume of their fibrillar centers which strongly suggest an ultradian rhythmicity. These results are different from those obtained in studies of superior cervical ganglion neurons, in which nucleoli and nucleolar components followed a circadian rhythm with peak values during daily periods of darkness. Although the nucleoli of these 2 kinds of neurons involved in autonomic nervous system reactions do not show the same behavioural patterns, the present data bring to light a new example of circadian fluctuation in nucleoli and describes their organization in this respect.

Circadian rhythm of nucleoli in rat superior cervical ganglion neurons: the two types of fibrillar centres and their quantitative relationship with the nucleolar organizing regions.

A quantitative stereological analysis was undertaken in nucleoli of rat superior cervical ganglion neurons. In this model, two types of fibrillar centres were observed: (1) small-type fibrillar centres were observed during the light span; and (2) a single large-type fibrillar centre occurred during the dark span near the smaller ones. The present data showed that the drastic increase in the mean volume of fibrillar centres during the dark span involved the occurrence and the progressive enlargement of one single large-type fibrillar centre and a marked rise in the number of small-type centres from 18 +/- 2 to 74 +/- 5 at 1500 h and 0100 h corresponding to light and dark spans, respectively. The total number of these small-type fibrillar centres per nucleolus increased with the total volume whereas their unit volume remained unchanged. This enabled us to establish some relationship with nucleolar organizing regions (NORs).

Three-dimensional distribution of Ag-NOR proteins in rat superior cervical ganglia nucleoli according to circadian rhythm.

A three-dimensional reconstruction of the distribution of Ag-NOR proteins in nucleoli of sympathetic neurons of a rat killed during the dark period of its light-dark cycle was compared with previously reported analyses on the three-dimensional distribution of fibrillar centers, the high-resolution localization of these proteins, and the morphometric results. The domain occupied by these proteins appeared to far exceed that of the fibrillar centers and included the dense fibrillar RNP component. In the present material this component in turn provided partial bridging between the units consisting of the fibrillar centers plus their surrounding dense fibrillar component.

Chronobiological studies on the nucleolus.

Chronobiological studies on the nucleolus were performed, using stereological analysis at the electron microscopic level, on different cell types in permanent interphasic state, in rats submitted to various lighting regimen. During the dark span (12L/12D): (1) in sympathetic neurons of superior cervical ganglion circadian changes in nucleolar organization were characterized by an increase in volumes of the nucleolus and each of its components, namely, fibrillar centres, dense fibrillar, granular and vacuolar components; (2) concerning the fibrillar centres, regarded as the interphasic counterpart of nucleolar organizing regions (NORs), the most striking fact, only observed in sympathetic neurons, is the occurrence of a single large-type fibrillar centre, accompanied by small-type fibrillar centres which are present throughout the 24-hr period; (3) the overall increase in volume of fibrillar centres was shown to correspond to a marked drop in the number (up to 4 fold) of small-type fibrillar centres, the unit volume of which (0.01 mum3) remaining unchanged over the 24-hr period and to an increase in size of a large-type fibrillar centre, the volume of which is 100 fold greater than the latter and (4) cytochemical studies showed that the Ag-NOR proteins exhibit a marked increase in amount, suggesting a circadian rhythmicity of these nucleolar proteins. These results, discussed in the light of our current understanding of the nucleolus, briefly summarized in this paper, suggest that the circadian rhythm of the nucleolus and of its components is correlated with circadian rhythms in both transcriptional activity and processing of preribosomes. Analogies between sympathetic neurons and the two other cell types studied, namely, chromaffin cells of adrenal medulla and vagal sensory neurons of nodose ganglion, led to the conclusion that rhythmicity is a fundamental characteristic of the nuclear structure devoted to ribosome biogenesis. Attention is focused on the superior cervical ganglion in which amplitude of nucleolar rhythms are greater and in which fibrillar centres exhibit a particular pattern. These results are discussed with regard to the role played by this sympathetic paravertebral ganglion which is known to regulate circadian rhythmic activities of the rat pineal gland. The persistence of these nucleolar rhythms in continuous lighting, as demonstrated in sympathetic superior cervical ganglion neurons, provide evidence that they are endogenously generated. The intrinsic factors underlying these rhythms in morpho-functional organization of the nucleolus are yet unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus. An ultrastructural and stereological study.

Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Group of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus under-goes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.

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.