Integrative and comparative genomic analyses identify clinically relevant pulmonary carcinoid groups and unveil the supra-carcinoids.

The worldwide incidence of pulmonary carcinoids is increasing, but little is known about their molecular characteristics. Through machine learning and multi-omics factor analysis, we compare and contrast the genomic profiles of 116 pulmonary carcinoids (including 35 atypical), 75 large-cell neuroendocrine carcinomas (LCNEC), and 66 small-cell lung cancers. Here we report that the integrative analyses on 257 lung neuroendocrine neoplasms stratify atypical carcinoids into two prognostic groups with a 10-year overall survival of 88% and 27%, respectively. We identify therapeutically relevant molecular groups of pulmonary carcinoids, suggesting DLL3 and the immune system as candidate therapeutic targets; we confirm the value of OTP expression levels for the prognosis and diagnosis of these diseases, and we unveil the group of supra-carcinoids. This group comprises samples with carcinoid-like morphology yet the molecular and clinical features of the deadly LCNEC, further supporting the previously proposed molecular link between the low- and high-grade lung neuroendocrine neoplasms.

Differential expression of telomerase reverse transcriptase (hTERT) in lung tumours.

Human telomerase reverse transcriptase is a ribonucleoprotein that synthesises telomeric sequences, which decrease at each cell division. In cancer cells, its activity is linked to telomere maintenance leading to unlimited cellular proliferation and immortality. To evaluate the prognostic value of the catalytic subunit telomerase reverse transcriptase (hTERT), we analysed its expression by immunohistochemistry in 122 formalin-fixed lung tumours including 42 squamous cell carcinoma (SCC), 43 adenocarcinoma (ADC), 19 basaloid carcinoma (BC) and 18 small-cell lung carcinoma (SCLC) in comparison with detection of hTERT mRNA by in situ hybridisation and relative telomerase activity by TRAP assay in a subset of tumours. We observed a high concordance between hTERT protein expression and detection of hTERT mRNA and telomerase activity. Telomerase expression varied according to histology (P=0.0002) being significantly lower in ADC than in SCC, BC and SCLC (P<0.0001). Adenocarcinoma and SCC exhibited either a nuclear or a nucleolar staining in contrast with a diffuse nuclear staining observed in most BC and all SCLC (P=0.01). In stage I NSCLC telomerase expression was lower than in other stages (P=0.04), and a nucleolar staining was correlated with a short survival (P=0.03). We concluded that telomerase expression and pattern are distinctive among histopathological classes of lung cancer and convey prognostic influence.

A simplified cytomegalovirus pp65 antigenemia assay procedure.

A simplified cytomegalovirus (CMV) pp65 antigenemia assay using a one-step erythrocyte lysis, fixation and permeabilization process was compared with a standard protocol, the CMV CINAkit (Argene Biosoft) assay. The results were comparable, both quantitatively and qualitatively. The new method saves time. It also provides flexibility because the cell suspension can be stored so that test completion can be deferred if so desired.

Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity.

In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.

TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement.

TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

Comparison of seven bio- and chemiluminescent reagents for in situ detection of antigens and nucleic acids.

Bio- and chemiluminescence have proved sensitive enough to compete with chromogenic and radioisotopic tracers for in situ detection. However, they must also provide a discriminant morphological analysis of the specific signal. We have tested seven bio- or chemiluminescent reagents for tissue antigen and nucleic acid detection by immunocytochemistry (ICC) or in situ hybridization (ISH). They were based on luminescent detection of peroxidase, alkaline phosphatase, beta-galactosidase or xanthine oxidase. We also explored whether high molecular weight polymers could increase the spatial definition of the photon emission. An ICCD camera was used to collect the light signal provided by immunolabelling of endothelial cells and by ISH of human papilloma virus on cell smears. Among the enzyme-luminescent substrate combinations tested, the enhanced luminol chemiluminescence (ECL) gave the best resolution of the specific signal. The other systems were mainly hampered by a high diffusion of the reaction product over the tissue section. Unfortunately, in this case, the high molecular weight polymers tested were inefficient. However, the addition of polyvinylalcohol (PVA) or polyvinylpyrrolidone (PVP) significantly improved respectively the definition and intensity of ECL photon emission. We demonstrate that chemiluminescence gives a morphological resolution allowing histological examination. The extension of this new application, now depends on physicochemical adaptation of chemiluminescent reagents to the constraints of tissue detection.

F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence.

We propose a general solution to the problem of using antibodies originating in the same species for double immunohistochemical labeling. It relies on the use of a two-step protocol in which a secondary polyclonal monovalent F(ab) antibody present in the first step blocks access in the second of the secondary antibody to the primary antibody, which is continuously present from the first step. The monovalence of the F(ab) fragment eliminates the possibility of its linking the primary antibody from the second step. We designed two efficiency tests to explore the limits of the method by the very sensitive chemiluminescent system applied to sections of human pituitary tissue. They confirmed both the validity of the method and the necessity of adapting working conditions to obtain a complete lack of interference.

Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry.

The breakthrough of chemiluminescence in the field of solution immunoassays and transfer membranes prompted us to explore whether a light-based detection system could provide a gain in sensitivity over chromogenic and FITC markers for nucleic acid and protein detection on histological preparations. A Hamamatsu device and an enhanced chemiluminescence (ECL) luminol substrate of the peroxidase were used to detect epithelial and endothelial components by immunohistochemistry (IHC) and for in situ hybridization (ISH) of papilloma virus DNA. The accuracy of the signal was compared to that obtained with DAB-peroxidase, silver-enhanced DAB-peroxidase, NBT-BCIP-alkaline phosphatase, and FITC. Our results demonstrated the feasibility and high sensitivity of luminescence detection for histological preparations. In part due to the ultrasensitive videocamera and photon-counting imaging, interpretable and reproducible results were obtained within counting times shorter than 5 min, and with dilutions of the primary antibodies 100- to 10,000-fold greater than those used for chromogenic and FITC reactions. As for ISH, with identical concentrations of the HPV 18 DNA probe on HeLa cells, labeling was apparent by luminescence but undetectable with the chromogen. The morphological resolution allowed a discriminatory analysis of the signal. Therefore, at the light microscopic level, enhanced chemiluminescence offers an appealing alternative to FITC and chromogenic markers for detection and quantification of low-concentration molecules.

Ultrastructural localization of the major components of the extracellular matrix in normal rat nerve.

In this study we determined the ultrastructural distribution of the various components of the extracellular matrix (laminin, fibronectin, Type I, III, and IV collagens) of the normal peripheral nerve in adult rat. The localization of these macromolecules was investigated in basement membranes as well as in different areas of epi-, peri-, and endoneurium, by use of a pre-embedding immunoperoxidase method.

[Hyperventilation syndrome: current advances]. / Syndrome d'hyperventilation: mise au point.

The hyperventilation syndrome has been described for half a century but clearly remains underdiagnosed. Its acute manifestation is easily diagnosed ("the tip of the iceberg") but its recognition in numerous subtle forms requires a particular degree of alertness on the part of the clinician ("the hidden part of the iceberg"). The incidence of this syndrome in the general population varies according to different authors as between 6-11% and may mimic diverse organic disorders. The physiological consequences of hyperventilation are reviewed as well as their contribution in the clinical picture. The aetiology of the syndrome and its links with organic pathology or psychiatric disturbances continues to be debated. Is hyperventilation the expression of abnormal respiratory function or a preferred manifestation of anxiety? This article discusses and reviews the variety of tests which enable the presumptive diagnosis to be confirmed. The response to the proposed treatments is generally excellent when one takes account of the numerous possible options. These include comportmental therapies such as respiratory re-education, the utilisation of betablockers and psychotrophic drugs or psychotherapy.

[Contribution of polysomnography in the assessment of patients with chronic obstructive bronchopneumopathy]. / Apport de la polysomnographie à la mise au point des malades atteints d'une bronchopneumopathie chronique obstructive (BPCO).

15 COPD patients underwent a polysomnographic study demonstrating poor quality of sleep, a mean of SAO2 of 88.8 +/- 3.9% and a apneic-hypopnea index (AHI) of 5.7 +/- 11.8. AHI was higher in sleep stages I and II than in REM sleep. SAO2 showed a progressive drop when going from an awake stage to REM sleep. Respiratory events responsible for the most important desaturation where mostly observed in REM sleep and corresponded in 8 patients to obstructive events (overlap syndrome). The lower mean SAO2 in REM probably explains the best the importance in desaturation related to the respiratory events (Hb dissociation curve). Ear oximetry recordings however interesting are not able to quantify and recognise correctly the respiratory events. Therefore a polysomnographic study remains necessary in order to diagnose adequately the overlap syndrome.

Quantitative image analysis with densitometry for immunohistochemistry and autoradiography of receptor binding sites--methodological considerations.

Major technical progress in the development of computer-based image analysis has made possible the entry of autoradiography and immunohistochemistry into a new era where quantification by densitometry has become easily accessible. Autoradiography could become quantitative and displayed adequate reproducibility with the help of emulsion-coated films and the use of scales of standards of known radioactivity exposed and analyzed in parallel to the tissue sections. Immunohistochemistry after revelation by a color-based enzymatic technique can also become quantitative, providing that standardization of the crucial steps of the procedure and calibration through a parallel treatment of a scale of antigen standards can be ensured. Such an approach is described here in the rat with reference to tyrosine hydroxylase (TH), the main synthesizing enzyme for catecholamines, and with dopamine (DA) itself, a catecholaminergic neurotransmitter. The different parts of the procedure, which can influence the results, such as the fixation of the animals by perfusion and the evaluation of the fluctuations via the calibration curve, are discussed in detail. Biological validation of the proposed procedure is described by reference to experiments already well documented biochemically, such as the induction effect of reserpine on TH in the rat locus coeruleus and the depleting effect of alpha-methyltyrosine (AMPT), a well-known blocker of TH activity, on rat striatal DA content. Finally the importance of restricting the measurements to the (pseudo)linear portion of the calibration curve is illustrated by the autoradiographic identification of the differential intrastriatal repartition of the dopaminergic D1 and D2 receptor sites, particularly the dual patch-matrix compartments.

[Value of monitoring of tracheal respiratory sounds in the diagnosis of nocturnal respiratory dysrhythmias]. / Intérêt du suivi des bruits respiratoires trachéaux dans le diagnostic de dysrythmies respiratoires nocturnes.

Twenty-six patients underwent a polysomnigraphic study allowing sleep staging and respiratory events scoring with the use of the oronasal flow, abdominal, thoracic and total displacement (Respitracet), and ear oximetry. Moreover the patients were also equipped with a tracheal microphone giving a power rectified envelope (sonospirogram). Eleven patients showed abnormal respiratory events that were scored by visual lecture using all respiratory parameters (excluding the sonospirogram) and were classified as obstructive central and mixed apneas-hypopneas. Periodic breathing was also appreciated. Detection of the same events was tried with the sonospirogram alone. The sonospirogram could accurately detect snoring and periodic breathing and finally central obstructive mixed apnea (the apneic index being well correlated: p less than 0.001 as well as the mean apnea duration: p less than 0.005). In contrast hypopneic events related to snoring could not be accurately appreciated. We conclude that a sonospirogram may be useful for the detection of abnormal respiratory events when used alone (screening) as when added to other respiratory signals.

A human monoclonal IgM with autoantibody activities against heparan sulphate and the mitotic spindle.

A monoclonal IgM kappa from a patient with Waldenström's macroglobulinaemia (IgM-Rod) was found to react at temperatures below 28 degrees C with all tissue basement membranes and the cell coat of non-haematopoietic cells. IgM-Rod antibody was directed against heparan sulphate side chains of heparan sulphate proteoglycans as shown by binding in a solid-phase ELISA to heparan sulphate glycosaminoglycans but not to other purified subcomponents of the extracellular matrix; and by specific inhibition of the observed reactivity by heparitinase treatment. IgM-Rod showed cross-reactivity by indirect immunofluorescence with an as yet unidentified structure expressed in the nucleus during cell division and becoming associated with the mitotic spindle apparatus. The co-existence of both binding activities for heparan sulphate and nuclei determinants in the same IgM molecule was deduced from adsorption-elution experiments and from the inhibitory effect of a mouse monoclonal anti-idiotypic antibody directed against the paratope of IgM-Rod.

[The value of a visual score of dyspnea in the functional follow-up of asthma patients]. / Intérêt du score visuel de dyspnée dans le suivi fonctionnel des asthmatiques.

6 severe asthmatic patients were followed for 24 hours using a visual analogue score of dyspnoea and by studying the airway resistance. Their treatment consisted either of two inhalations of placebo or of two inhalations of fenoterol every 5 hours. It appeared that there was only a correlation between the dyspnoea score and respiratory function score for the largest variations of resistance induced by inhalation of sympathicomimetics. The inverse was that altered function offering only small fluctuations was not correlated with a sensation of dyspnoea.

Cyclic haemodynamic and arterial blood gas changes during Cheyne-Stokes breathing.

A 74-year-old patient presented with congestive heart failure and continuous periodic breathing. Left ventricular ejection fraction was 20% and the lung-to-brain circulation time was prolonged to 35 s. We report on the phasic changes of the patient's arterial blood gas tensions and on the periodic fluctuations of pulmonary artery pressures and cardiac output that we observed during Swan-Ganz catheterisation.

[Sleep studies in adults: interest in snoring disorders and the sleep apnea syndrome]. / Les études de sommeil chez l'adulte: intérêt dans la ronchopathie et dans le syndrome d'apnée du sommeil.

The authors review the concept of sleep apnea syndrome in adults. A description of a polysomnigraphic study is given. Personal results in a population of obese heavy snorers are summarized.