Pekka Laurila

Fluorescent antibodies and lectins stain intracellular structures in fixed cells treated with nonionic detergent.

Nonionic detergent (NP40) treatment of paraformaldehyde-fixed normal and SV40-transformed human fibroblasts resulted in intracellular penetration of two chosen fluorescent antibodies and Concanavalin A (Con A). After the detergent treatment nuclear SV40 T antigen, cytoplasmic fibronectin glycoprotein and Con A binding sites could be visualized in fluorescence microscopy. The lowest NP40 concentration which made fixed cells permeable was 0.05%. The morphology of cells was preserved better by this new method than by conventional fixation methods, such as acetone treatment. In scanning electron microscopy the surface of the fixed NP40-treated cells had only small rugosities and fine pores. The subsurface cytoskeleton especially was well preserved and had a more distinct fine structure. The improved morphology made it possible to detect a similar distribution of fibronectin and Con A binding sites in the perinuclear endoplasmic reticulum regions.

The HPV test has similar sensitivity but more overdiagnosis than the Pap test—A randomised health services study on cervical cancer screening in Finland

We compared test sensitivity (in terms of prevented cancers) and overdiagnosis (in terms of non‐progressive pre‐invasive lesions) between the human papillomavirus test (HPV test, Hybrid Capture 2) and the traditional Pap test in routine screening for cervical cancer. The design was a randomised (1:1) health services study in Finland with intake between 2003 and 2007. We estimated sensitivity by the incidence method within one screening round. Overdiagnosis was based on the rate of cervical intraepithelial Grade 3 (CIN3) lesions diagnosed at screen and during the following interval. Out of 203,788 randomised women 132,298 attended (65% in both study arms) and 600,753 person‐years accumulated among attenders up to the end of 2010. In all attenders, 34 invasive cervical cancers and 288 CIN3 lesions were diagnosed at screen or during the following interval. The interval cancer incidence was 2.5/105 person‐years (sensitivity 0.87) and 1.4 (sensitivity 0.93) in the HPV arm and Pap test arm, respectively. The rate of CIN3 lesions was 57.1 and 38.8, respectively. In conclusion, sensitivity of HPV testing was similar to that of Pap testing but caused more overdiagnosis. Therefore, implementation of HPV testing needs to be reconsidered especially in countries with well organised programmes.

Characterization of Storage Material in Cultured Fibroblasts by Specific Lectin Binding in Lysosomal Storage Diseases

Summary: The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure.In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for JV-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.Speculation: Staining of cultured cells with sugar-specific lectins may reveal interesting aspects on the pathogenesis of lysosomal storage phenomenon. In some instances, the method can be of use as an auxiliary technique in prenatal diagnosis of lysosomal storage diseases.

Intermediate filaments in enucleation of human fibroblasts

Abstract Enucleation of cultured human fibroblasts was used to study the interaction between the nucleus and the cytoskeletal intermediate filaments. In enucleated cells the filaments stained for immuno-fluorescence were seen as fibrillar cytoplasmic arrays similar to those of intact cells. Typical reorganization of the filaments into coiling bundles occurred in cells treated with vinblastine either before or after enucleation. The cytoplasmic stalk connecting the extruding nucleus to the rest of the cell contained filaments, whereas the karyoplasts lacked them. The present results indicate that the filaments are essential for the anchorage of the nucleus and that the weakest point in this system is between the nucleus and the intermediate filaments.

Expression of fibronectin matrix in cybrids of fibroblasts and epithelial cells

The pericellular fibronectin matrix of human fibroblasts is lost when they are fused with normal or malignant cells, which do not produce fibronectin matrix. In the present study we have investigated whether also the cytoplasmic fraction of fibronectin-negative HeLa or MDCK cells can cause this effect. Enucleated epithelial cells (cytoplasts) were therefore fused with norman human fibroblasts. Fibronectin expression of the resulting cytoplasmic hybrids (cybrids) was studied by the indirect immunofluorescence technique. Three hours after fusion cybrids formed between fibroblasts and enucleated epithelial cells showed fibronectin matrix expression clearly weaker than that seen on the intact fibroblasts. An accumulation of fibronectin matrix was observed in the cybrids, analogously to the intact fibroblasts, and already 12-24 h after fusion the cybrids showed a fibronectin matrix expression similar to that of the fibroblasts. No fibronectin matrix was detected in the epithelial cells or their cytoplast. Our results indicate that cytoplasmic factors from epithelial cells are able to cause an initial suppression in the formation of fibronectin matrix. However, the cytoplasts are not capable of causing a long-term effect on the fibroblasts.

Loss of pericellular fibronectin matrix from heterokaryons of normal human fibroblasts and epithelial cells

Abstract The expression of fibronectin in heterokaryons of normal human fibroblasts and normal or malignant epithelial cells was studied by indirect immunofluorescence microscopy. Fibroblasts and their homokaryons showed a characteristic pericellular fibronectin matrix, whereas both normal (MDCK) and malignant (HeLa) epithelial cells, and their homokaryons, lacked such a matrix. The fibroblast homokaryons also showed a typical strong, perinuclear cytoplasmic, fibronectin-specific fluorescence. This was much weaker or absent in the MDCK and HeLa cells and their homokaryons. When human fibroblasts were fused with either normal or malignant epithelial cells, no pericellular matrix-like, fibronectin-specific fluorescence could be seen in the heterokaryons. Interestingly, however, a distinct intracellular fluorescence was seen in the heterokaryons, indicating continued production of fibronectin. The results of the present study indicate that both malignant and normal epithelial cells, which do not deposit fibronectin matrix, can cause its loss in heterokaryons with fibroblasts. Thus, discontinued fibronectin matrix formation does not point exclusively to malignancy, but may also reflect the state of differentiation of the parental cells.

Stillbirths and maternal antibodies to Chlamydia trachomatis. A new EIA test for serology

Objective. The relationship between stillbirths and infections during pregnancy.