Biomedical subjects

E Visse

Publications and source records attributed to E Visse.

6 recordsLinked to original sources

PubMed2001

Computerized image analysis as a tool to quantify infiltrating leukocytes: a comparison between high- and low-magnification images.

The purpose of the present study was to establish a rapid and reproducible method for quantification of tissue-infiltrating leukocytes using computerized image analysis. To achieve this, the staining procedure, the image acquisition, and the image analysis method were optimized. Because of the adaptive features of the human eye, computerized image analysis is more sensitive to variations in staining compared with manual image analysis. To minimize variations in staining, an automated immunostainer was used. With a digital scanner camera, low-magnification images could be sampled at high resolution, thus making it possible to analyze larger tissue sections. Image analysis was performed by color thresholding of the digital images based on values of hue, saturation, and intensity color mode, which we consider superior to the red, green, and blue color mode for analysis of most histological stains. To evaluate the method, we compared computerized analysis of images with a x100 or a x12.5 magnification to assess leukocytes infiltrating rat brain tumors after peripheral immunizations with tumor cells genetically modified to express rat interferon-gamma (IFN-gamma) or medium controls. The results generated by both methods correlated well and did not show any significant differences. The method allows efficient and reproducible processing of large tissue sections that is less time-consuming than conventional methods and can be performed with standard equipment and software.(J Histochem Cytochem 49:1073-1079, 2001)

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PubMed2000

Immunohistochemical analysis of glioma-infiltrating leucocytes after peripheral therapeutic immunization with interferon-gamma-transfected glioma cells.

We have shown previously that rejection of preinduced rat brain tumours is possible following therapeutic immunizations with interferon-gamma (IFN gamma)-transfected glioma cells (N32-IFN gamma). In the present study we have used the same model to evaluate whether quantitative differences in tumour-infiltrating lymphocytes can be detected between animals receiving therapeutic immunizations with either IFN gamma-transfected glioma cells, wild-type glioma cells or no treatment. Since leucocyte transpedesis into the tumour can be anticipated to depend on the state of vascularization, we have mapped the development of microvessels in the tumour in parallel with the leucocyte infiltration. Our results show that microvessels start to form at day 7 and then gradually increase in number and size, indicating the establishment of an extensive vascularization by day 24. Leucocyte infiltration displays a biphasic pattern after tumour grafting. We have therefore studied the infiltration kinetics after an early immunization (1 day after intracerebral isografting) and compared the effects with those of a late immunization (10 days after intracerebral isografting) with N32-IFN gamma or wild-type N32. Our results show (1) an early infiltration of granulocytes 3 days after isografting; (2) a T-cell-receptor-positive (TCR+) T-cell infiltration starting on day 10; (3) a macrophage infiltration starting on day 13; (4) a CD8+ cell infiltration starting on day 13. The proportions of TCR+ T cells, CD8+ cells and natural killer cells differs significantly between animals immunized with N32-IFN gamma and those receiving wild-type N32, when analysed 14 days after immunization at day 10. This difference can only be detected when animals are immunized at later stages of tumour growth. We propose that this could depend on an early-immunization-independent leucocyte infiltration during tumour establishment. This has to be considered when evaluating studies of leucocyte infiltration in experimental tumours.

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PubMed1996

Cure of established, intracerebral rat gliomas induced by therapeutic immunizations with tumor cells and purified APC or adjuvant IFN-gamma treatment.

We have previously reported that immunizations with mutagen-induced immunogenic variants of a weakly immunogenic rat glioma could protect against isografts of the original tumor cells. In this study we show that prolonged survival and cures of rats with established gliomas in their brains can be achieved by therapeutic immunizations with tumor cell mutants, combined with in vitro and in vivo interferon (IFN)-gamma (adjuvant) treatment, or tumor cells admixed with semipurified syngeneic dendritic cells. Cure of rats with established intracerebral gliomas was possible when immunizations were initiated up to 5 days after intracerebral isografting of original tumor cells. Unexpectedly, immunizations combined with in vitro and in vivo IFN-gamma treatment or with admixed semipurified dendritic cells equalized the immunogenic potential of the original tumor cells and that of mutagen-induced immunogenic cell variants (tum-). This demonstrates that effective immunizations against a weakly immunogenic brain tumor can be achieved by different adjuvant concepts. The therapeutic effect of immunizations with tumor cells admixed with semipurified dendritic cells was highly significant in female rats, whereas only occasional cures and prolonged survival were recorded in male rats. The overall results show that therapeutic immunizations can indeed be effective against an established and growing intracerebral tumor.

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PubMed1995

Increased proportion of CD8+ tumor responsive T cells after immunization with tum- versus tum+ rat glioma.

Previously established immunogenic (tum-) clones of an ENU (ethyl-N-nitrosourea)-induced rat glioma, N32, were compared to the original tumor concerning their capacity to induce T lymphocyte responses after in vivo immunization and in vitro restimulation of responder spleen cells in mixed lymphocyte tumor culture (MLTC) assays. Quite unexpectedly, original N32 (tum+) in vivo primed spleen cells proliferated to the same extent in vitro in response to tum+ stimulator cells as did tum- in vivo primed spleen cells. However, flow-cytometric analysis of parallel cultures showed a greatly increased proportion of CD3+CD8+ lymphocytes in the proliferating responder cell population from tum- immunized hosts, contrary to a CD3+CD4+ lymphocyte dominance after tum+ immunization. Although the original tum+ N32 tumor cells are not capable of inducing a clearly demonstrable isograft rejection response, they induce a strong T cell response readily detectable in MLTC assays. We propose that the increased CD8+ lymphocyte proliferation could be an essential feature of the isograft rejection response induced by tum- tumor variants. Possible mechanisms of the augmented CD8+ T cell response are discussed.

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PubMed1993

Immunization with mutagen-treated (tum-) cells causes rejection of nonimmunogenic rat glioma isografts.

The ethyl-N-nitrosourea-induced rat glioma N32 was treated with the mutagenic compound N-methyl-N'-nitro-N-nitrosoguanidine and the surviving cells cloned by limited dilution. Out of 20 clones tested 8 did not produce tumors subcutaneously even after challenge doses 3 log units above the minimal tumor dose for N32. All of 5 clones grew in a retarded manner intracerebrally but produced tumors in some animals. Preimmunizations with three of the rejected clones (tum-) gave protection against subcutaneous and intracerebral isografts of the unmutated N32. This effect could be enhanced if the cells used for immunizations were pretreated with interferon gamma (IFN gamma) for 48 h. If immunizations were started subsequent to challenge, only immunization with one of two tested tum- clones pretreated with IFN gamma induced significant rejection against intracerebral N32 isografts. Both N32 and its tum- clones were MHC class I positive and MHC class II negative. IFN gamma treatment enhanced the MHC class I expression with 20%-90% on the tum- clones and with 40% on N32. MHC class II expression could be induced on N32 cells after 7 days of IFN gamma treatment but not on any of the tum- clones tested. We conclude that the enhancing effect of IFN gamma treatment on tumor isograft rejection may depend on up-regulation of MHC class I but not of MHC class II. This investigation demonstrates that it is possible to induce rejection of weakly immunogenic intracerebral brain tumors by immunization with selected highly immunogenic tumor cell mutants. In conjunction with relevant cytokines, the cross-protective effect of these tum- variants might be further enhanced and serve as a model for immunotherapy against malignant human brain tumors.

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PubMedDate not supplied

Regression of intracerebral rat glioma isografts by therapeutic subcutaneous immunization with interferon-gamma, interleukin-7, or B7-1-transfected tumor cells.

Progress in the definition of the roles of various costimulators and cytokines in determining the type and height of immune responses has made it important to explore genetically altered tumor cells expressing such molecules for therapeutic immunizations. We have studied the effect of therapeutic subcutaneous (s.c.) immunizations on the growth of preexisting intracerebral brain tumor isografts in the rat. Transfectant glioma cell clones expressing either rat interferon-gamma (IFN-gamma), rat interleukin-7 (IL-7), or rat B7-1 were selected. After irradiation (80 Gy) the clones were used for immunization (administered in up to four s.c. doses in a hind leg over 14-day intervals starting 1 day after the intracranial isografting of the parental tumor). Significant growth inhibition of the intracerebral parental tumors was induced by transfectants expressing IFN-gamma and IL-7, respectively. The strongest effect was observed with IFN-gamma-expressing cells, resulting in cures in 37% of the males and in 100% of the females. Immunization with IL-7 had a similar, strong initial effect, with significantly prolonged survival in the majority of the rats but a lower final cure rate (survival for >150 days). The B7-1-expressing tumor clones induced cures in seven of eight female rats; however, no cures were seen in the male rats. It was also shown that the B7-1-expressing cells were themselves strongly immunogenic in female rats, requiring high cell numbers to result in a progressively growing tumor upon s.c. isografting; this was not the case in male rats. As a whole, the results imply that despite the unfavorable location of intracerebral tumors, therapeutic s.c. immunizations with certain types of genetically altered tumor cells can induce complete regressions with permanent survival and without gross neurological or other apparent signs of brain damage. The present results demonstrate complete regressions when immunizations are initiated shortly after intracranial isografting, when the intracerebral tumor is small.

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