Classification of cervical smears with discordance between the cytologic and/or histologic ratings

The problems of diagnostic variability between certified cytotechnologists was studied. Three cytology laboratories submitted a total of 28 cervical smears that had a discordance between the cytologic and/or histologic ratings. Eight independent cytotechnologists provided blind readings on each slide, expressed as "absence of cervical intraepithelial neoplasia (CIN)" to "CIN III." The median rating was absence of CIN or CIN I for 8 slides, CIN II for 5 and CIN III for 15. With a kappa value greater than 0 reflecting agreement beyond chance expectation and a value of 0.40 indicating fair agreement, the kappa value for 8 X 28 ratings was 0.36 (P = .0001), with a 90% confidence interval (CI) between 0.34 and 0.37. The kappa value was 0.14 (P = .10), with a 90% CI between 0.10 and 0.18, on a subsample of nine smears with two or more positive cytology diagnoses but a negative histology. Sixteen of the 28 slides represented cases of histologically proven cancer. Treating cytologic diagnoses of CIN II and CIN III as positive, the sensitivity of the cytologist with reference to histology varied between 71% and 86% while the specificity ranged from 18% to 62%. The positive predictive value was 1/2.5 to 1/1 and the negative predictive value was 1/6 to 1/1. The predictive power (true positives/false positives) ranged from 1.0 to 2.2. The cytodiagnosis of these cervical smears from cases of discordance thus exhibited limited reliability. Standardization of the relevant cytologic knowledge and its routine application is needed to improve the level of performance.     

Determination of tissue-type plasminogen-activator mRNA in human and non-human cell lines by dot-blot hybridization.

A labelled cDNA clone was used in DNA-RNA hybridization on nitrocellulose filter paper (dot-blot technique) to detect and quantify mRNA for endogenous tissue plasminogen activator (PA) in cell extracts and samples of RNA purification runs. Although, for detection purposes, the assay was less sensitive than translation in Xenopus oocytes, it was at least as reliable and much more convenient for the purpose of quantitative determination. In particular, the technique was used to study the kinetics of PA mRNA formation in a human melanoma cell line (Bowes) after exposure to the tumour promoter 12-O-tetradecanoylphorbol 13-acetate (TPA). Incubation of the cells with TPA resulted in a 15-20-fold increase in cellular PA mRNA content. The effect was time- and dose-dependent: the increase in PA-specific mRNA was clearly visible as early as 4 h after initiation of TPA treatment in Bowes cells. It was blocked completely by pretreatment of the cells with actinomycin D, indicating that TPA caused enhancement of synthesis of PA mRNA rather than inhibition of PA mRNA degradation. The use of the nitrocellulose dot-blot technique also revealed that two non-human cell lines produce mRNAs which cross-react with the human PA mRNA, namely the mouse melanoma cell line B16 and the rat brain-tumour cell line, RT4-71-1. TPA was found to exert similar stimulatory effects on the synthesis of mRNAs in these cell lines as in Bowes cells.     

Influence of carbohydrate side chains on activity of tissue-type plasminogen activator

When messenger RNA (mRNA) from both untreated and phorbol ester-treated melanoma cells is translated in simple reticulocyte lysates, tissue-type plasminogen activator can be immunoprecipitated by an affinity-purified antibody as a approximately 52,000 mol wt protein, with no detectable biological (plasminogen activating) activity. When the reticulocyte lysate system is supplemented with a preparation of microsomal membranes, biological activity becomes detectable and a 63,000 mol wt protein can be immunoprecipitated with the same antibody. Furthermore, when natural tissue-type plasminogen activator (mol wt approximately equal to 70,000) is incubated with different glycosidases, distinct alterations in the electrophoretic mobility of the molecules are observed, together with alterations in the level of biological activity. While treatment with neuraminidase and beta-galactosidase caused decreases in activity, alpha-mannosidase caused an increase. These results suggest that the carbohydrate part of the molecule can influence its biological behavior.     

Increased phospholipid synthesis in the stimulated rat and human pancreas

Stimulation of the exocrine pancreas is associated with marked changes in pancreatic phospholipid metabolism. It has been previously established that de novo synthesis of phospholipids constitutes part of this "phospholipid effect". This study has demonstrated that in vitro stimulation of the rat pancreas utilising bethanecol and pancreozymin results in increased incorporation of labelled glucose into phosphatidyl inositol and, to a lesser extent, other phospholipids, suggesting increased de novo synthesis of these compounds. However, secretin which is believed to act via a different intracellular pathway, did not exert such an effect. The relevance of this animal model is indicated by the demonstration of increased incorporation of labelled glucose into phospholipids of human pancreas stimulated in vitro by bethanecol or sincalide (the active carboxy terminal octapeptide of pancreozymin).     

Is the meniscus of the knee joint a fibrocartilage?

A histological analysis of the structure of intact knee joint menisci was carried out in adult dogs. By means of specific histochemical methods for the connective tissue and cartilage, it was found that the meniscus as a whole does not have a unique structure. The anterior and posterior horns are populated by round chondroid cells encircled by abundant interstitial substance and branched wavy connective fibers; blood vessels are present. The outer third of the meniscus is constituted of cross bundles of connective fibers, fibrocytes and spindle-like areas of loose connective tissue with blood vessels. The inner avascular two thirds of the meniscus are filled with parallel circumferentially oriented fascicles of connective fibers, ovally elongated chondroid cells, and a small quantity of chondroid interstitial substance. In some menisci, in the inner two thirds of the body, there are isles of typical cartilage, which show metachromasia of the beta type and rarely of the gamma type. The occurrence and way of the manifestation of cartilage are of an individual character. The structural duality of the knee meniscus is accounted for by its functional duality manifested in offering resistance to the forces of traction and pressure, the latter ones favoring the process of evolution of tissue from connective, through chondroid, to cartilaginous.     

Rubella virus interference and interferon production

An interference technique for the titration of rubella virus on primary rabbit kidney (PRK) cells using either vaccinia or vesicular stomatitis virus (VSV) as challenge is described.Rubella virus passed and propagated on PRK-cells interfered with a challenge dose of either vaccinia virus or VSV given on the 7th day after inoculation. Concomitantly, interferon was demonstrated in these cultures in amounts up to 100 units per ml.The same strain of rubella virus passed on a continuous rabbit kidney cell line (RK13) induced no interferon in PRK-cells. Yet it interfered equally well with a challenge of vaccinia virus or VSV.Under experimental conditions where no interferon could be detected, rubella virus growth inhibited the one-cycle multiplication of poliovirus-RNA, suggesting that interference resides at the intracellular level.Under the described experimental conditions the multiplication of rubella virus in PRK-cells was not inhibited by an amount of interferon 20 times higher than that necessary to inhibit vaccinia virus cytopathic effect in the same cell system.Aspirant of the Belgian N.F.W.O.Navorsingsstagiair of the Belgian N.F.W.O.     

Silencing of the MEG3 gene promoted anti-cancer activity and drug sensitivity in glioma

Aberrant expression of MEG3 has been shown in various cancers. The purpose of this study is to evaluate the effect of MEG3 on glioma cells and the use of potential chemotherapeutics in glioma by modulating MEG3 expression. Cell viability, migration and chemosensitivity were assayed. Cell death was evaluated in MEG3 overexpressing and MEG3 suppressed cells. MEG3 expression was compared in patient-derived glioma cells concerning IDH1 mutation and WHO grades. Silencing of MEG3 inhibited cell proliferation and reduced cell migration while overexpression of MEG3 promoted proliferation in glioma cells. MEG3 inhibition improved the chemosensitivity of glioma cells to 5-fluorouracil (5FU) but not to navitoclax. On the other hand, there is no significant effect of MEG3 expression on temozolamide (TMZ) treatment which is a standard chemotherapeutic agent in glioma. Suppression of the MEG3 gene in patient-derived oligodendroglioma cells also showed the same effect whereas glioblastoma cell proliferation and chemosensitivity were not affected by MEG3 inhibition. Further, as a possible cell death mechanism of action apoptosis was investigated. Although MEG3 is a widely known tumour suppressor gene and its loss is associated with several cancer types, here we reported that MEG3 inhibition can be used for improving the efficiency of known chemotherapeutic drug sensitivity. We propose that the level of MEG3 should be evaluated in the treatment of different glioma subtypes that are resistant to effective drugs to increase the potential effective drug applications.     

Neurochemical effects ofl-pyroglutamic acid

The effect ofl-pyroglutamic acid, a metabolite that accumulates in pyroglutamic aciduria, on different neurochemical parameters was investigated in adult male Wistar rats. Glutamate binding, adenylate cyclase activity and G protein coupling to adenylate cyclase were assayed in the presence of the acid.l-pyroglutamic acid decreased Na⁺-dependent and Na⁺-independent glutamate binding Basal and GMP-PNP stimulated adenylate cyclase activity were not affected by the acid. Furthermore, rats received unilateral intrastriatal injections of 10–300 nmol of bufferedl-pyroglutamic acid. Vehicle (0.25 M Tris-Cl, pH 7.35–7.4) was injected into the contralateral striatum. Neurotoxic damage was assessed seven days after the injection by histological examination and by weighing both cerebral hemispheres. No difference in histology or weight could be identified between hemispheres. These results suggest that, although capable of interfering with glutamate binding, pyroglutamate did not cause a major lesion in the present model of neurotoxicity.