The effects of irradiation on cell migration from glioblastoma multiforme biopsy spheroids.

BACKGROUND: Glioblastoma multiforme (GBM) is a primary brain tumour with a very poor prognosis despite aggressive multi-modality treatment. This pre-clinical experimental study focuses on the effect of irradiation on three-dimensional glioma biopsy spheroids in vitro using an outgrowth assay to evaluate cell survival and migrational capacity of the glioma cells. MATERIALS AND METHODS: Tumour tissue of 16 patients with high-grade glioma and two GBM cell lines were used for spheroid preparation. Outgrowth and cell density were the parameters chosen to evaluate cell cytotoxicity and migrational capacity after irradiation (20 Gy and 4 x 5 Gy). RESULTS: Radiation inhibited outgrowth of cell line spheroids, but not of the biopsy spheroids. All biopsy and cell line spheroids showed a significantly lower cell number (95 vs. 24 cells/0.25 mm2) in the outgrowth area after irradiation. CONCLUSION: Irradiation has a cytotoxic effect in GBM biopsy spheroids but it hardly affects cell migration. No correlation was found between patient survival and cell migration nor with cytotoxicity.

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.

Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood.

Cytoplasmic granules from activated natural killer (NK) and cytotoxic T lymphocytes (CTL) contain a pore-forming protein, perforin, and several homologous serine proteinases called granzymes. Expression of these proteins correlates with the cytolytic potential of cytotoxic lymphocytes. Using a panel of MoAbs specific for human granzyme A and B, respectively, expression of these proteinases in non-pathological lymphoid tissue and peripheral blood lymphocyte (PBL) subpopulations was investigated. Using immunohistochemistry and double stainings, the phenotype of granzyme-expressing cells in lymphoid tissue was investigated. Granzyme-positive cells were detected in all lymphoid tissues tested. No large differences in the number and distribution between granzyme A- and granzyme B-positive cells were observed. The highest number of positive cells was located in the red pulp of the spleen. Significant numbers were detected in tonsil, lymph nodes, liver and thymus. Low numbers were present in the lamina propria of non-inflamed stomach, small intestine and colon. Phenotypic analysis and cell sorting showed that most of the granzyme-positive cells in lymphoid tissue and PBL consisted of CD3-CD16+CD56+ lymphocytes. Hardly any granzyme-positive CD3+CD8+ CTL were present in peripheral blood. The synthesis of granzyme A as well as B by both CD3+CD16+CD56+ and CD3+CD8+ cells in peripheral blood was increased upon IL-2 stimulation. These results indicate that in normal lymphoid tissue the predominant cytolytic cell population is formed by the NK cells, and activated CTL are rare.

The contribution of immunocytochemistry in diagnostic cytology. Comparison and evaluation with immunohistology.

This study reports on the use and evaluation of immunocytochemistry in diagnostic cytology by correlation with (immuno)histology. The cytologic material was collected during a period of 3 years. Sixty-four patients were selected and studied retrospectively. The reactivity of a number of polyclonal and monoclonal antibodies was investigated and applied to a diverse group of tumors. Marker expression on routine cytologic and histologic material was compared and the use of immunocytochemistry in diagnosis was evaluated. Immunocytochemistry proved to be an easy and valuable technique for the identification and classification of tumor cells, and there was a good concordance with immunohistology. A discrepancy in marker expression was found in 10% of the slides that were investigated. Immunocytochemistry led to a misdiagnosis in only four cases; however, in these cases cytology alone would not have led to the correct diagnosis. The causes for the discrepancies are discussed. It is estimated that immunocytochemistry contributed to the diagnosis of approximately 50% of the cases, supplying either additional or essential information. A wide variety of markers can be applied reliably on cytologic preparations. The use of panels of antibodies is mandatory.

Combined immuno- and non-radioactive hybridocytochemistry on cells and tissue sections: influence of fixation, enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences.

Conditions for combination of DNA in situ hybridization, using biotinylated DNA probes, with immunohistochemistry were investigated on cryostat sections, cytological preparations, and paraffin sections. We found that cryostat sections and cytological preparations are suitable for in situ hybridization of target DNA after fixation in acetone, methanol, ethanol, or Carnoy without further proteinase pretreatment. Acetone is also very suitable for immunostaining of cell surface or cytoskeleton antigens. We therefore performed combined immunoenzyme and in situ hybridization staining using this fixative. The best results were obtained when immunoperoxidase staining with diaminobenzidine/H2O2 was followed directly by in situ hybridization. In addition to immunoperoxidase, alkaline phosphatase-antialkaline phosphatase (APAAP) staining with naphthol ASBI phosphate and New Fuchsin as a substrate could be used. In most instances, detection of the biotinylated hybrid with a streptavidin-biotinylated polyalkaline phosphatase method using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate as the substrate was preferable. The double stainings were studied on the following test models: (a) frozen tonsil sections: cell surface antigens (pan T) and ribosomal DNA; (b) frozen genital condyloma sections; cytokeratins and human papillomavirus type 6 + 11 (HPV-6/11) DNA; (c) CaSKi cells: cytokeratins and HPV-16 DNA; (d) infected fetal lung fibroblasts: vimentin and cytomegalovirus (CMV) DNA. An adapted procedure was followed on routinely formaldehye-fixed and paraffin-embedded condyloma tissue. Immunoperoxidase staining for papilloma virus capsid antigen could be combined with DNA in situ hybridization with HPV-6/11 DNA. In this model, however, the accessibility of the target DNA had to be improved by enzyme treatment after the immunostaining and before starting the in situ hybridization.

Immunohistochemistry in bone marrow diagnosis. Value of a panel of monoclonal antibodies on routinely processed bone marrow biopsies.

To see if immunohistochemistry can be used on routinely processed bone marrow biopsies for diagnostic purposes, 73 biopsy specimens, fixed in sublimate-formaldehyde, decalcified in an acetic acid-formaldehyde mixture, and embedded in paraffin, were studied with a panel of antibodies. The specimens included "normal," lymphomatous, and myeloproliferative disorders as well as some biopsies with metastatic carcinoma. The results show that the different cell lines and their localization in the bone marrow can be easily identified and quantitative and qualitative changes can be assessed. Megakaryopoiesis is identified with Factor VIII-related antigen and Ulex europaeus agglutinin (UEA); myelopoiesis stains with MT-1, elastase, Leu M-1, LN-2, LN-3, HECA 452, and 115D8; and in myeloproliferative conditions, myeloblasts and promyelocytes stained with leukocyte common antigen (LCA). Erythroid cells stained with UEA, glycophorin A, and LN-1. Lymphocytes were marked with LCA, MB-2, and LN-2. Plasma cells were stained best with immunoglobulin light chain antisera; only occasional reactivity with LCA and 115D8 was observed. Carcinomas all reacted with MB-2; occasional reactivity with 115D8, HECA 452, LN-1, LN-2, MT-1, and Ber H-2 was seen. A small panel of selected antibodies, such as UEA, Leu M-1, and LCA, and the immunoglobulin light chain antisera can be very helpful in bone marrow diagnosis and would cover most indications.

Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma.

The occurrence and coexpression of the cytoskeletal proteins vimentin and cytokeratins were studied in malignant mesotheliomas and pulmonary carcinomas. For this purpose a double immunoenzyme staining with monoclonal antibodies was developed which made it possible to visualize vimentin and cytokeratins simultaneously within the same cell. A clear distinction between stromal cells (vimentin only) and tumour cells was also obtained. A total of 12 mesotheliomas (six mixed type and six epithelioid type) and 13 carcinomas (eight adenocarcinomas and five large cell undifferentiated carcinomas) were studied. The results revealed a clear difference between mesotheliomas and adenocarcinomas: 11 of 12 mesotheliomas showed coexpression of vimentin and cytokeratins in at least 50% of the tumour cells, while in seven of the eight adenocarcinomas none or only a few cells could be seen with this coexpression. In the undifferentiated large cell carcinomas three of five expressed both components, but in less than 25% of the cells. It is concluded that a reliable double immunoenzyme staining of vimentin and cytokeratins can be used as an additional means to distinguish malignant mesothelioma from pulmonary adenocarcinoma.

Influence of fixation and decalcification on the immunohistochemical staining of cell-specific markers in paraffin-embedded human bone biopsies.

A number of fixation and decalcification procedures were evaluated to determine their suitability for immunohistochemistry on trephine samples of bone marrow after paraffin embedding. In particular, the immunoreactivity of antigens characteristic for various hematopoietic cell lines (immunoglobulin heavy and light chains for plasmacytoid cells; elastase for neutrophil myeloid cells; lysozyme, alpha-1-antitrypsin and alpha-1-antichymotrypsin for hystiocytic cells; leukocyte common antigen for lymphocytes; hemoglobin and glycophorin A for erythroid cells; Factor VIII-related antigen for thrombocytoid cells) as well as some antigens specific for epithelial tumors (CEA, 115D8, and keratin) were investigated. Fixation in a mercuric chloride-formaldehyde mixture followed by decalcification in acetic acid-formaldehyde-saline proved to be the best procedure for antigen preservation and retention of morphologic detail. Moreover, there is no need of trypsinization when using this procedure. The only exception was Factor VIII-related antigen in megakaryocytes, which was best demonstrated in trypsin-digested sections of formalin-fixed and acetic acid-decalcified biopsies.

Membranous glomerulonephritis in the mouse.

Glomerulonephritis was induced in C57. B110 mice by a single injection of rabbit IgG against homologous, pronase-digested, renal tubular antigens. The heterologous phase was characterized by a transient increase of glomerular permeability with fixation of rabbit IgG to the capillary walls, in a linear or fine-granular pattern, and to the brush borders of the proximal tubuli. The autologous phase was marked by the immune response to the injected protein, during which subepithelial immune deposits, consisting of mouse IgG1, rabbit IgG, and mouse C3 developed. Small amounts were still present at 1 year after the injection of antiserum. The antibody response of the mice correlated with the development and resolution of the deposits. None of the mice developed a nephrotic syndrome. Control mice treated with normal rabbit IgG did not show immune deposits in their kidneys at any stage despite a comparable antibody response to rabbit IgG. Immunoelectronmicroscopy showed that the rabbit antibodies fixed directly to an antigen in the cell membrane of the glomerular visceral epithelium. It seems, therefore, likely that in situ formation of subepithelial immune complexes occurred in the autologous phase by fixation of mouse immunoglobulins to rabbit IgG already present in the glomerular wall.