Understanding anaphors in story dialogue.

Previous research has identified two processes that play an important role in anaphor resolution. An activation process increases the accessibility of an anaphor's referent; a suppression process diminishes the accessibility of its nonreferents. In this study, we examined how these processes operate when reference to two story characters shifts rapidly, as it does in story dialogue. Dialogue raises interesting questions about how the antecedent of an anaphor becomes the most activated entity in the reader's discourse model. Do readers suppress an anaphor's nonreferent even though that same entity is likely to be the referent of a subsequent anaphor? Are activation and suppression processes triggered by the anaphor itself or by cues that signal a change of speaker? We found that an anaphor's antecedent is activated differently in dialogue than it is elsewhere in a narrative. Our results suggest that readers use knowledge about the structure of dialogue to anticipate the referent of an upcoming anaphor.

T-cell evaluation in patients with colon cancer: dinitrochlorobenzene skin testing versus plasma levels of sIL-2r and sCD8.

BACKGROUND: Developing reliable methods to test the T-cell system may be important in the treatment of colon cancer patients with 5-fluorouracil/levamisole. In a pilot study we explored whether DNCB (dinitrochlorobenzene) skin testing correlated with plasma levels of soluble interleukin-2 receptor (sIL-2r) and soluble CD8 (sCD8) and, secondly, whether the application of DNCB had any influence on the production of sIL-2r and sCD8. METHODS: In 10 patients with advanced colon cancer and in 10 healthy volunteers, plasma levels of sIL-2r and sCD8 were measured before and 10 days after the application of 2 mg DNCB on the inner side of the forearm. RESULTS: As expected, colon cancer patients showed a depressed immune system compared to healthy volunteers (DNCB skin test: P = .005, sIL2r [medians 700 vs 295, P = .002], sCD8 [medians 158 vs 90, P = .03], M-W test). The plasma levels for sIL-2r and sCD8 were significantly lower in the skin-positive cases (P = .01 and P = .03, M-W test). However, a large overlap in plasma levels could be observed between the two skin categories. DNCB had no influence on the production of sIL-2r and sCD8; median change skin-negative and skin-positive -10 vs +25, P = .14, respectively; 48 vs 0, P = .32 (M-W test). CONCLUSIONS: DNCB skin testing and plasma levels of sIL-2r and sCD8 seem to be equally useful in evaluating the T-cell system and can be used simultaneously.

Abnormal A-type lamin organization in a human lung carcinoma cell line.

We have studied the expression of lamins A and C (A-type lamins) in a lung carcinoma cell line using type-specific monoclonal antibodies. Using immunofluorescence and immunoblotting studies it was noted that several irregularities in lamin expression exist in the cell line GLC-A1, derived from an adenocarcinoma. First, the expression of the A-type lamins was lower than in other adenocarcinoma cell lines of the lung. Also the ratio between lamins A and C proteins was 1:8 instead of the 1:1 ratio seen in the other cell lines. Northern blotting confirmed the altered level of A-type lamin expression. Secondly, an abnormal localization of lamin A was observed. Intensely fluorescing lamin A aggregates were observed in the nucleus, rather than the typical perinuclear staining pattern. Confocal scanning laser microscopy revealed that the lamin A aggregates were indeed present throughout the internal nucleus. When these cells were extracted with Triton X-100 the nucleoplasmic aggregates disappeared, which indicates that the A-type lamins are not properly incorporated into the lamina. The A-type lamins in other cell lines derived from adenocarcinomas remained present in the nuclear periphery after extraction with the non-ionic detergent. Immunoblotting studies of the Triton X-100 soluble and insoluble fractions showed that lamin A and an apparently truncated product, which was detected with the lamin A antibody, were present in the insoluble fraction of GLC-A1. This truncated product is partly Triton X-100 soluble since it was also detected in the detergent soluble fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of steroids on brain lipocortin immunoreactivity.

LCT-1, LCT-2 and LCT-5 were assessed in uninjured rats and rats subjected to a cortical freezing injury or middle cerebral artery (MCA) occlusion. Apart from animals receiving no treatment, other uninjured or injured animals received methylprednisolone (2 or 30 mg/kg) or the 21-aminosteroid U-74389F (10 mg/kg) one day and 2 hours before killing. The animals were killed by decapitation 1 hour after the freezing injury or the MCA occlusion and the area containing the lesion was removed and frozen in Freon. Frozen sections were treated with rabbit polyclonal anti-LCT antibody; binding of antibody was visualized by horseradish peroxidase-conjugated swine antirabbit antibody. Without steroid pretreatment, in the uninjured brain LCT immunoreactivity was absent in the greater part of the brain, except in sporadic microglia. In steroid-pretreated animals and in the freezing lesion of both pretreated and untreated animals there was extensive immunostaining; in the freezing lesion it may be due to passage of systemic LCT across the impaired blood-brain barrier in the lesion. The cellular elements showing immunostaining were meningeal cells, neurons, ependyma, choroid plexus, oligodendroglia and capillary endothelium. It implies that also in the brain the steroid effect is consistent with LCT formation.

The effect of steroid treatment on lipocortin immunoreactivity of rat brain.

Lipocortin-1, lipocortin-2 and lipocortin-5 were immunohistochemically assessed in rats. Apart from animals receiving no treatment, other animals received pretreatment with methylprednisolone, or the 21-aminosteroid U-74389F. Whereas Hpocortin immunoreactivity was absent in the greater part of the brain in animals not pretreated with steroid (except in sporadic microglial cells and choroid plexus), there was obvious immunostaining of parenchymatous elements in steroid pretreated animals. In the steroid pretreated animals lipocortin immunoreactivity of the brain tissue may indicate local formation of lipocortin under the influence of steroids that had entered the tissue. The cellular elements which showed immunostaining included meningeal cells, neurones, ependyma, oligodendroglia and capillary endotheHum.

Our approach towards developing a specific tumour-targeted MRI contrast agent for the brain.

This review presents various aspects of the technological development, and their assessment in the design of a contrast agent for MRI, tailored to visualise tumours in the brain. First, it was demonstrated that magnetite as a contrast agent exhibited a much stronger relaxivity than gadolinium. The prepared magnetite particles bound to dextran, were also shown to be of appropriate size by electron microscopy. After their intravenous injection into rats with blood-brain barrier disruption, the lesion was strongly enhanced by T2-shortening. Furthermore, monoclonal antibodies directed against small cell lung carcinoma, proved to be able to penetrate into tumours, which had been raised by implantation of the small cell lung carcinoma cells into the brains of nude rats. As to the essential step, it was demonstrated in vitro that magnetite particles coupled to monoclonal antibodies by the biotin-streptavidin binding, could be bound to the target cells of the antibody, changing the relaxation rates of the latter. Finally it could be shown in vitro that an alternative approach, using lymphocytes to be targeted to tumour cells, also proved feasible, in that these lymphocytes could be labelled with magnetite that had been incorporated into liposomes. Further developments will be the in vivo assessment of the acquired progress in experimental animals, before clinical application is warranted.

Distribution of endotracheally instilled surfactant protein SP-C in lung-lavaged rabbits.

In lung-lavaged surfactant-deficient rabbits (n = 6) requiring artificial ventilation, porcine surfactant was instilled endotracheally. This resulted in improvement of lung function so that the animals could be weaned off artificial ventilation. The animals were killed 4 1/2 h after surfactant administration and the porcine surfactant protein was localized in the lung with a MAb. We found surfactant protein in all lobes of the lung but the distribution was not homogeneous. Surfactant protein C was found in less than 15% of the alveolar spaces and in less than 1% of the bronchi.

Proliferation rate of colonic mucosa in normal subjects and patients with colonic neoplasms: a refined immunohistochemical method.

An increased colonic epithelial proliferation rate and an increase of the cryptal proliferative zone are probable markers of increased susceptibility to colonic cancer. In this study an immunohistochemical method using 5-bromo-deoxyuridine (BrdUrd) to measure the proliferation rate of colonic mucosa in vitro was used. Fresh endoscopic colonic biopsy specimens were incubated with BrdUrd and then processed for immunohistochemistry using a monoclonal antibody. Essential procedures with respect to the equal distribution of nuclei stained with BrdUrd in the biopsy specimens proved to be the cutting of the specimens before incubation and the use of a microwave oven at the beginning of incubation. The use of the procedure of the running average showed that 12 length cut crypts are sufficient to determine reliably the proliferation rate, expressed as the labelling index (LI). This was determined in the biopsy specimens of 10 subjects without organic colonic disease, eight patients with adenomatous colonic polyps, and in six patients with (recent) colonic carcinoma. Mean LI in the controls was significantly lower than in patients with colonic polyps and in those with colon cancer. It is concluded that this method is promising for screening persons at risk for colon cancer and will be of great potential in performing dietary intervention studies in these subjects.

Characterization of a human small cell lung carcinoma cell line with acquired resistance to cis-diamminedichloroplatinum(II) in vitro.

A 6.4-fold cis-diamminedichloroplatinum(II) (CDDP) resistant human small cell lung carcinoma cell line (GLC4-CDDP) was developed to study acquired CDDP resistance in vitro. Compared to the sensitive cell line (GLC4), the GLC4-CDDP showed an increase in doubling time and a decrease in cloning efficiency, cellular size, double minutes per cell, cellular protein, and nuclear protein content. While a complete cross-resistance for tetraplatin and a partial cross-resistance for doxorubicin, melphalan, cadmium chloride, carboplatin, and cis-dichloro-trans-dihydroxo-cis-bis(isoprolylamine)platinum (IV) (resistance factor, respectively,4.0,5.8,2.1,1.5,2.9) was found, no cross-resistance for vincristine was found. In the GLC4-CDDP line in comparison to the GLC4 line, glutathione and total amount of sulfhydryl compounds was significantly increased, while glutathione S-transferase and glutathione reductase was the same. The platinum content in cells and nuclei was lower in the resistant line, but after correction for cellular protein or volume no difference was found. The amount of platinum bound to DNA was significantly lower in the GLC4-CDDP line. After a 1-h incubation with CDDP, the amount of Pt-GG adducts was the same and the amount of interstrand cross-links was reduced in the GLC4-CDDP line as compared to GLC4. In conclusion, in the GLC4-CDDP line the phenotype and genotype are changed and various mechanisms, such as decreased Pt-DNA binding, elevated glutathione, and reduced interstrand cross-links, play a role in the development of the CDDP resistance.

Two small cell lung cancer cell lines established from rigid bronchoscope biopsies.

Two new, good growing cell lines (GLC-8, GLC-11) have been established from biopsies of small cell lung cancer (SCLC). Tumor biopsies were procured by rigid bronchoscopy from tumor recurrences at the site of the primary lesions. Both tumors were clinically resistant to chemotherapy. Cytogenetic analysis revealed deletions in the short arm of chromosome 3. GLC-8 shows amplification of N-myc. Both cell lines show SCLC differentiations; neurosecretory granules were present and the SCLC related hormones dopa-decarboxylase and creatine kinase were elevated. Both cell lines behave as so-called 'classic' SCLC cell lines.

Double immunohistochemical demonstration of antigen expression and DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections.

In this study a double immunohistochemical staining procedure is described for the simultaneous visualization of antigen expressing cells and replicating cells. Cell surface antigen expression was marked with a monoclonal antibody against I a (His 19) or a monoclonal antibody against a membrane component of the cells of the monocyte-macrophage lineage (ED2). Replicating cells were detected by the incorporation of 5-bromodeoxyuridine. The method was applied sequentially. On frozen sections two peroxidase labeled reagents were used with two different substrates yielding a red and a dark-blue black reaction product. On plastic-embedded sections a peroxidase and an alkaline phosphatase labeled reagent were applied resulting in a brown and a blue reaction product.

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections.

In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.

Intermediate filament expression in small cell lung cancer; poor correlation to in vitro data.

A panel of monoclonal antibodies, detecting different intermediate sized filament proteins, was prospectively applied on all specimens derived from S.C.L.C. patients attending our clinic in 1985. Reactivity with the antibodies was subsequently correlated to clinical data. The results indicate a heterogeneous pattern of reactivity of the assessed antibodies. However this heterogeneity is not a straightforward extension of the intermediate sized filament expression in SCLC cell lines.

Immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine in frozen and plastic embedded sections.

The application of an immunohistochemical method in the detection of replicating cells, that have incorporated 5-bromodeoxyuridine (BrdUrd), was studied on frozen and plastic embedded sections of different rat tissues. Hydrolysis conditions employed in the Feulgen procedure are essential in making the incorporated BrdUrd accessible to the monoclonal anti-BrdUrd antibodies. To demonstrate the incorporated BrdUrd in plastic embedded sections a subsequent etching with xylene and digestion with protease is necessary. Data obtained with this method are completely comparable with those found by the tritiated thymidine method. In comparison with the thymidine method, the BrdUrd method is much less time consuming and does not require precautions in working with radioactivity. The BrdUrd-method enables a more precise localization as is especially shown in the plastic embedded sections.

Differential expression of intermediate filament proteins distinguishes classic from variant small-cell lung cancer cell lines.

The expression of intermediate filament proteins in classic and variant-type small-cell lung carcinoma (SCLC) cell lines was studied using immunocytochemical techniques, two-dimensional gel electrophoresis and immunoblotting assays. Classic SCLC cell lines contain cytokeratin proteins but no neurofilaments. In contrast, variant cell lines do not contain detectable amounts of cytokeratins but partly express neurofilaments and vimentin. These results explain apparent discrepancies on the intermediate filament content of SCLC described in the recent literature. The application of antibodies to fresh biopsy specimens of SCLC may in the future allow the identification of the variant type of cells in clinical SCLC specimens and may have a major impact on therapeutic strategy and prognosis in these patients.

Morphologic, immunologic, enzymehistochemical and chromosomal analysis of a cell line derived from Hodgkin's disease. Evidence for a B-cell origin of Sternberg-Reed cells.

Cell lines derived from Hodgkin's disease may provide a clue to the nature of Sternberg-Reed cells. In the current study, the establishment of an Epstein-Barr-virus-negative lymphoblastoid cell line, derived from the pleural fluid of a patient with the nodular sclerosis type of Hodgkin's disease, is described. The morphologic and immunologic cell marker findings indicate that this cell line is derived from Sternberg-Reed cells. The immunologic findings and a chromosomal analysis are in agreement with a B-lymphocyte origin of these cultured cells. Extrapolation of the results to Hodgkin's disease in vivo would indicate that Hodgkin's disease, like most non-Hodgkin's lymphomas, is the result of B-cell proliferation.

Serum-dependent "cannibalism" and autodestruction in cultures of human small cell carcinoma of the lung.

In cell lines of human small cell carcinoma of the lung (SCCL) and in all subclones of one of the cell lines, cells were observed which completely interiorized other cells, leading to death of the interiorized cells and sometimes to complete autodestruction of the cultures. This phenomenon, which we have called "cannibalism," is also observed in fresh tumor biopsies from SCCL patients. Cannibalistic cells appeared to be of SCCL origin. "Cannibalism" is never observed in serum-free cultures but can be reinduced by serum exposure. It is likely that "cannibalism" may contribute to the frequent failure to establish SCCL cell lines in serum-containing medium. The potential to induce autodestruction of tumor cells in SCCL patients by as yet unknown serum factor(s) may be of therapeutic value.

Identification of three indolic compounds in a pigmented-melanoma cell-culture supernatant by gas chromatography-mass spectrometry.

In the supernatant of melanoma cell culture SK mel 25 three indolic compounds - 5-hydroxy-6-methoxyindole, 5-hydroxy-6-methoxyindolyl-2-carboxylic, and 6-hydroxy-5-methoxyindolyl-2-carboxylic acids - have been identified. The supernatants were extracted with ethyl acetate, derivatized, and analyzed by gas chromatography-mass spectrometry. The significance of this finding is discussed.