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L de Leij

Publications and source records attributed to L de Leij.

At least 55 records · Page 3Linked to original sources
PubMed1993

Refinement of an indirect immunotoxin assay of monoclonal antibodies recognising the human small cell lung cancer cluster 2 antigen.

Monoclonal antibodies (Mabs) from the Second International Workshop on Small Cell Lung Cancer (SCLC) Antigens that recognise the cluster 2 SCLC-associated antigen mediated potent and selective cytotoxic effects in an indirect assay of immunotoxin cytotoxicity. In this assay, the NCI-H69 cell line was treated with each Mab at 4 degrees C, washed to remove unbound Mab, and then incubated at 37 degrees C in the presence of a fixed concentration, 1 x 10(-8) M, of the screening agent, sheep anti-mouse IgG-ricin A chain. The use of a fixed high concentration of screening agent led to a 300-fold overestimate of the potency of a cluster 2-directed immunotoxin, MOC-31-ricin A chain. In contrast, when the concentration of the screening agent was identical to the Mab concentration, a precise match to immunotoxin potency was obtained. MOC-31-ricin A chain selectivity inhibited the incorporation of [3H]leucine by the NCI-H69, SW2 and GLC-8 SCLC cell lines by 50% at a concentration between 3 x 10(-11) M and 3 x 10(-10) M, and by the NCI-H125 lung adenocarcinoma cell line at 7 x 10(-11) M, but exerted no selective toxic effects upon human lung and non-lung tumour cell lines lacking surface expression of the cluster 2 antigen.

Antibodies, Monoclonal↗
PubMed1992

Direct visualisation and quantification of cellular cytotoxicity using two colour flourescence.

A fluorescence method is described for the evaluation of cell death induced by cellular cytolytic activity. A green fluorescent membrane dye, D275, was used to label various target cell lines and propidium iodide (PI) uptake was used to assay cell death. Natural killer (NK), lymphokine activated killer (LAK) as well as targeted T cell mediated cellular cytotoxicity were quantified using the fluorescence method and compared to results obtained with the 51chromium (51Cr) release assay. A good correlation was found after an assay period of 4-8 h indicating that the fluorescence method is a reliable alternative to the 51Cr release assay.

Carbocyanines↗
PubMed1992

Increased production of antigen-specific B lymphocytes during in vitro immunization using carrier-specific T helper hybridomas.

An in vitro method to increase the production of hapten-specific antibody-forming B cells (AFC) using a carrier-specific T helper hybridoma and murine splenocytes is described. Naive splenocytes (6 x 10(6)/ml) are cultured in vitro in the presence of a hapten-carrier conjugate (DNP.OVA) and OVA-specific T helper hybridomas (0.5 x 10(6)/ml). After 4-5 days in vitro immunization (IVI), the maximum number of DNP-specific AFC were found using a spot-ELISA with twice the number of IgM positive cells as IgG positive AFC. The presence of antigen in the form of a hapten-carrier complex and the use of a carrier-specific Th hybridoma resulted in more hapten-specific AFC than when neither antigen nor Th hybridoma were present or when antigen alone or T help alone were used. Also when the hapten was conjugated to a carrier not recognised by the carrier-specific Th hybridoma there were considerably fewer (less than 50%) hapten specific AFC formed. When in vivo primed splenocytes (DNP) were boosted in vitro (IVB) under the same conditions as for IVI most hapten-specific AFC were found on day 4 and both anti-DNP IgM and IgG AFC were increased relative to IVI. Again most AFC were found when hapten was bound to the relevant carrier. In conclusion, carrier-specific T hybridomas can be used in an in vitro immunization procedure with naive or primed splenocytes to increase the frequency of anti-hapten AFC. This method offers an improvement over the current in vitro immunization procedures for the production of monoclonal antibodies.

Animals↗
PubMed1992

Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC).

Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.

Amino Acid Sequence↗
PubMed1992

Dextran-magnetite particles: contrast-enhanced MRI of blood-brain barrier disruption in a rat model.

Dextran-magnetite particles (DMP) were studied for their use as a MR contrast agent to visualize lesions with a blood-brain barrier (bbb) disruption. A freezing injury to the rat cerebral cortex was used as a model of bbb disruption. The biodistribution of iv-injected DMP was studied using atomic absorption spectrophotometry, electron microscopy, and MRI. One hour after injection, focal accumulation of the particles in capillary endothelial cells could be demonstrated in the freezing lesion. Despite the observation that the relaxivity of DMP in vivo appears to be less well pronounced than that in vitro, the MR imaging studies show that DMP can be used to visualize bbb disruption with adequate contrast.

Animals↗
PubMed1992

Peripheral blood lymphocyte number and phenotype prior to therapy correlate with response in subcutaneously applied rIL-2 therapy of renal cell carcinoma.

The phenotype of peripheral blood lymphocytes of 27 renal cell carcinoma patients before and at the end of subcutaneously given rIL-2 therapy was determined by two colour flow cytometry. Therapy induced changes in peripheral blood leucocyte composition and phenotypes were comparable to those reported for intravenously given rIL-2. The present paper shows a correlation between the 'activation status' of the patient before therapy and eventual response.

Adult↗
PubMed1991

Neuroectodermal tumors of the peripheral and the central nervous system share neuroendocrine N-CAM-related antigens with small cell lung carcinomas.

The current study describes the presence of neuroendocrine antigens of peripheral and central neural tumors using eight monoclonal antibodies raised to small cell lung carcinoma (SCLC), which recognize "neural/neuroendocrine" or "neural" antigens, as defined by their reaction pattern in normal tissues and tumors. At least five of them recognize different epitopes of the neural cell adhesion molecule (N-CAM). It was found that all of 12 neuroblastomas, 2 ganglioneuroblastomas and 4 ganglioneuromas as well as 23 central primitive neuroectodermal tumors, 13 astrocytomas and 4 ependymomas share "neural/neuroendocrine" antigens (as defined by the anti-N-CAM antibodies Moc-1, -21, -32, -52 and -191) with SCLC. The "neural/neuroendocrine" antigen defined by Moc-171 was also found in all peripheral tumors, but only in further differentiated central tumors. Non-N-CAM related "neural" antigens (as defined by Moc-51 and -172) were found only in better-differentiated peripheral and central tumors, but they could be demonstrated in all three medulloblastoma cell lines studied. In addition, the antigen defined by Moc-51 was demonstrated in an immunoblot of a neuroblastoma cell line. Antibodies recognizing "epithelial" antigens of SCLC and other epithelia and their tumors (Moc-31 and -181) were non-reactive. It was concluded that these findings give further support for a relation between neural and neuroendocrine tumors and that some of the antibodies may be useful for the detection of differentiation in neural tumors. Antibodies with an "epithelial" recognition pattern may serve to distinguish neural from neuroendocrine tumors.

Adolescent↗
PubMed1990

A study of human small-cell lung carcinoma (hSCLC) cell lines with different sensitivities to detect relevant mechanisms of cisplatin (CDDP) resistance.

The cisplatin(CDDP)-resistant cell line GLC4-CDDP shows a variety of differences from the parent line GLC4. The aim of this study was to determine which of the observed changes correlated with the degree of resistance and was therefore relevant to the phenomenon of CDDP resistance. For these experiments we used cells of the sensitive hSCLC cell line GLC4 and the in vitro-acquired CDDP-resistant sublines GLC4-CDDP3 and GLC4-CDDP11, with a resistance factor (RF) of 3 and 11 respectively for CDDP and of 1.8 and 7.4 respectively for carboplatin. Carboplatin was used, in addition to CDDP in seeking relevant mechanisms. No consistency was found between the RF and the growth pattern or antigen expression, cellular volume, doubling time, cellular or nuclear platinum (Pt) content or the level of Pt-non-histone chromatin protein (NHCP) binding. A correlation was found between the RF and the level of glutathione (GSH), and a trend was found for the level of Pt-DNA binding, Pt-GG adduct content and the amount of interstrand cross-links (ISC). These changes might therefore be relevant for the development of resistance. These findings are compatible with a GSH-induced reduction of the amount of reactive Pt in the resistant cell, resulting in a lower net platination and toxic Pt-DNA adduct formation.

Antibodies, Monoclonal↗
PubMed1990

Passage of DMP across a disrupted BBB in the context of antibody-mediated MR imaging of brain metastases.

To study the possible application of monoclonal antibody/dextran-magnetite conjugates in specific MR imaging of brain metastases, both components of these conjugates were tested for their ability to penetrate the endothelium during conditions of local blood-brain barrier (BBB) impairment. The passage of dextran-magnetite particles (DMP) across a disrupted blood-brain barrier was studied in a freezing lesion model using electron microscopy (EM) and MR imaging. One hour after i.v. injection, focal accumulation of DMP in capillary endothelial cells within the freezing lesion was shown by EM. In parallel with this, MR imaging indicated a strong contrast enhancement in the lesion. EM observations showed that the particles were still present in the endothelial cells four and eight hours after injection. The passage of an anti-small cell lung cancer (SCLC) monoclonal antibody across the endothelium of intracerebrally xenografted human SCLC was studied using immunohistological techniques. It was found that passage across endothelial cell occurred in the tumor within four hours after injection.

Animals↗
PubMed1989

Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow-up.

Mechanisms for resistance were studied in three classic type, human small cell lung cancer cell lines, GLC14, GLC16, and GLC19, that were established from one patient during clinical follow-up. Clinically the tumor changed from sensitive (GLC14) to completely resistant to (chemo)therapy (GLC19) during this period. The stain with JSB-1 antibody, detecting the Mr 170,000 multidrug resistance associated glycoprotein, was most pronounced in GLC16 and absent in GLC19. Intracellular Adriamycin (Adr) concentrations were decreased in GLC16 and GLC19 versus GLC14. Glutathione levels were 12.9, 15.5, and 16.6 micrograms/mg protein; total sulfhydryl groups were 36.5, 45.7, and 48.8 micrograms/mg protein; and glutathione S-transferase activity was 13, 29, and 43 nmol I-chloro-2,4-dinitrobenzene/min/mg protein for GLC14, GLC16, and GLC19, respectively. Incubation with DL-buthionine-S,R-sulfoximine increased Adr and cisplatin induced cytotoxicity, whereas X-ray induced cytotoxicity remained the same. Catalase activity increased from 0.88 to 1.73 to 3.83 mumol H2O2/min/mg protein in, respectively, GLC14, GLC16, and GLC19. Compared to GLC14 and GLC16, Adr induced a higher amount of DNA strand breaks in GLC19. In none of the three cell lines could Adr induced DNA strand breaks be repaired. X-ray induced a comparable amount of DNA strand breaks in all three cell lines but all cell lines were capable of repairing the X-ray induced DNA strand breaks within 90 min. It is concluded that a number of different mechanisms are operative and that some but not all of the observed changes in mechanisms for drug resistance in these lines correlate with the clinical data.

Antineoplastic Agents↗
PubMed1989

Characteristics of a multicopy gene family predominantly consisting of processed pseudogenes.

The monoclonal antibody MOC-32 detected a 40 kDa protein in Western blot analysis. Immunological screening of an expression library of human SCLC cells with MOC-32 led to the isolation of overlapping cDNA clones. One of these, cHD4, was 1.0 kbp long and of about the same size as its corresponding mRNA. Preceded by an in phase stop codon, an open reading frame of 885 bp was present in cHD4 and a translational product of only 33 kDa could be calculated. Biochemical and immunological analysis established the relationship between the 40 kDa antigen and the isolated coding sequences and resolved the apparent discrepancy between the calculated molecular weight and the observed electrophoretic mobility. Nucleotide sequence comparison of cHD4 to the EMBL database revealed that cHD4 was nearly identical to a sequence claimed to encode a laminin binding protein. Southern blot and nucleotide sequence analysis indicated the presence of multiple copies of the gene in the human genome. At least five of these appeared to represent processed pseudogenes.

Amino Acid Sequence↗
PubMed1989

Heterogeneity of Wilms' tumour blastema. An immunohistological study.

Antigen expression in fourteen cases of Wilms' tumour was assessed with a panel of monoclonal antibodies. The panel included antibodies reactive with intermediate filament proteins and antibodies reactive with membrane markers originally reported to be associated with small cell lung carcinoma. The immunohistological findings in the tumours were compared to results obtained in adult and fetal kidney. The antigen profile in the blastemal tissue component of the tumours revealed both characteristics of embryonic tissue and signs of early epithelial differentiation. In addition, the histologically apparent transition between blastema and tubules was shown to be reflected in a concurrently occurring and gradual increase in the number of expressed epithelial antigens. Between different tumours, heterogeneity in the degree of epithelial differentiation in the blastema was found. In addition, one case showing foci of anaplasia proved to have an entirely different antigen profile when compared with the other thirteen, non-anaplastic cases. This result is discussed in relation to the different clinical behaviour of focally anaplastic tumours. It is concluded that immunohistology can confirm and extend the histological classification of Wilms' tumour. In addition, new subtypes may be identified in this way.

Antibodies, Monoclonal↗
PubMed1989

Expression of intermediate filament proteins in fetal and adult human lung tissues.

The expression patterns of intermediate filament proteins in fetal and normal or nonpathological adult human lung tissues are described using (chain-specific) monoclonal antibodies. In early stages of development (9-10 weeks and 25 weeks of gestation) only so-called simple cytokeratins such as cytokeratins 7 (minor amounts). 8, 18 and 19 are detected in bronchial epithelial cells. At later stages of development, the cytokeratin expression patterns become more complex. The number of bronchial cells positive for cytokeratin 7 increases, but basal cells in the bronchial epithelium remain negative. These latter cells show, however, expression of cytokeratin 14 in the third trimester of gestation. Developing alveolar epithelial cells express cytokeratins 7, 8, 18 and 19. In adult human bronchial epithelium cytokeratins 4 (varying amounts), 7, 8, 13 (minor amounts), 14, 18 and 19 can be detected, with the main expression of cytokeratins 7, 8, and 18 in columnar cells and the main expression of cytokeratin 14 in basal cells. Vimentin is detected in all mesenchymal tissues. In addition, fetal lung expresses vimentin in bronchial epithelium, however, to a lesser extent with increasing age, resulting in the expression of vimentin in only few scattered bronchial cells at birth. Also in adult bronchial epithelium the expression of vimentin is noticed in part of the basal and columnar epithelial cells. Desmin filaments, present in smooth muscle cells of the lung, appear to alter their protein structure with age. In early stages of development smooth muscle cells surrounding blood vessels are partly reactive with some cytokeratin antibodies and with a polyclonal desmin antibody. At week 9-10 and week 25 of gestation a monoclonal antibody to desmin, however, is not reactive with blood vessel smooth muscle cells but is only reactive with smooth muscle cells surrounding bronchi. With increasing age the reactivity of cytokeratin antibodies with smooth muscle cells in blood vessels decreases, while the reactivity with the monoclonal desmin antibody increases. Our results show that during differentiation profound changes in the intermediate filament expression patterns occur in the different cell types of the developing lung.

Antibodies, Monoclonal↗
PubMed1989

Clinical characterization of non-small-cell lung cancer tumors showing neuroendocrine differentiation features.

In most cases of small-cell lung carcinomas (SCLC) phenotypic features compatible with a neuroendocrine differentiation status can be identified by monoclonal (MOC) antibody-based immunohistological procedures. Similar features can be recognized only in a minority of non-SCLC tumors. During a period of 30 months, all diagnostic non-SCLC biopsies (141 cases) were prospectively analysed for the presence of markers indicative for neuroendocrine differentiation. In 31% of all cases, such a presence could be noticed. Neuroendocrine differentiation (50% to 100% positive-staining tumor cells) was recognized more frequently in adenocarcinoma when compared to large-cell and squamous-cell carcinoma (chi 2 = 9.31, 2 degrees of freedom, P less than 0.01). To investigate whether the clinical behavior of these "neuroendocrine" non-SCLC cases mimics SCLC, a multivariate analysis for prognostic factors was performed. Among other prognostic factors, biopsies containing more than 50% positive-staining tumor cells with the MOC antibody-1 (MOC-1) were recognized as negative prognostic factors.

Antibodies, Monoclonal↗
PubMed1988

Characterization of three small cell lung cancer cell lines established from one patient during longitudinal follow-up.

Three classic-type, small cell lung cancer cell lines (GLC-14, GLC-16, and GLC-19) have been established from one patient during longitudinal follow-up. During this period the tumor changed from sensitive to completely resistant to (chemo)therapy. A phenotypical and functional characterization of the different cell lines is given in combination with the matching clinical data. (a) The cell lines have been compared with the biopsies from which they were derived. There was a good match between the morphological, biochemical, and immunohistological findings in the cell lines as compared to those obtained in the biopsies. When the biopsy and cell line (GLC-14) obtained before the start of therapy were compared to the biopsies and cell lines (GLC-16 and GLC-19) acquired after the first and second reinduction therapy, respectively, no major changes could be observed. The only clear alteration was the loss of a neuroendocrine antigen (defined by monoclonal antibody MOC-51) in the posttherapy specimens. (b) The doxorubicin, melphalan, and etoposide sensitivity in vitro reflected the clinically observed development of resistance to treatment. The cell line (GLC-14) established before the start of therapy was more sensitive than the lines (GLC-16 and GLC-19) obtained after treatment. It is concluded that the cell lines described in this paper represent a well-characterized in vitro model in which the development of drug resistance in small cell lung cancer can be studied.

Antigens, Differentiation↗
PubMed1988

Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript.

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against alpha A-crystallin and identified the antigenic determinant for two of these antibodies. Applying limited-digestion methods, we show that the region spanning residues 158-168 of alpha A-crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated alpha Ains-crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the alpha A-crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.

Animals↗