Biomedical subjects

J C Tonn

Publications and source records attributed to J C Tonn.

At least 55 records · Page 3Linked to original sources

PubMed1999

Microglial/macrophage expression of interleukin 10 in human glioblastomas.

Interleukin 10 (IL-10) expression has been found to be correlated with the extent of malignancy in gliomas. In vitro, IL-10 increases proliferation and migratory capacity in human glioma cell lines. In this study, we localized the site of IL-10 synthesis in gliomas to cells of microglial origin. Biopsy specimens from 11 patients with malignant glioma were processed on native tissues and at early cell culture passages (0-4). IL-10 mRNA was analyzed by RT-PCR and in situ hybridization. Protein was quantitatively assessed by ELISA in cell culture supernatants, and cells expressing IL-10 were determined by a combination of immunohistochemistry for CD68 (specific for microglia/macrophage lineage) and IL-10 in situ hybridization. IL-10 mRNA decreased from passage 0 to 4 in all samples and was undetectable beyond passage 5. Such downregulation of mRNA leads to a steep decrease of IL-10 protein in culture supernatants (below detection level, 0.05 ng/ml, beyond passage 1). The combination of in situ hybridization for IL-10 and CD68 immunostaining revealed that only cells of the microglia/macrophage lineage produced IL-10 mRNA. Our results identify microglia/macrophage cells as the major source of IL-10 expression in gliomas which decreases markedly during early passages of primary cultures of human gliomas due to a progressive reduction of microglia/macrophages present.

Adult↗

PubMed1999

Effect of synthetic matrix-metalloproteinase inhibitors on invasive capacity and proliferation of human malignant gliomas in vitro.

Glioma invasion into the surrounding brain tissue is still a major obstacle for any therapeutical approach. As in other solid tumors, matrix-metalloproteases (MMPs) have been suggested as being involved. The aim of this study was to evaluate whether the use of MMP inhibitors to target the protease-mediated invasion process could be a feasible approach. Two human cell lines (U251 and GaMG) and surgical specimens of 6 patients with malignant gliomas were grown as monolayers and spheroid cultures respectively. MMP- and u-PA-mRNA expression was investigated by semi-quantitative RT-PCR. Invasion was studied in Matrigel-coated Boyden chamber transwell assays for monolayers and in confrontation cultures of tumor spheroids with fetal rat brain aggregates in the presence of the synthetic MMP inhibitors batimastat (BB-94) and marimastat (BB-2516). Cytotoxicity/cytostatic effects of high concentrations of both compounds were assessed by growth curves, MTT assays and flow cytometry in human glioma cell lines. Batimastat and marimastat revealed a cytostatic effect at high concentrations (above 1 microM) without cytotoxicity. Both MMP inhibitors effectively reduced glioma invasion in Boyden-chamber assays at low concentrations of 0.3 microM. In confrontation cultures, concentrations of 10 microM and above were necessary to reduce invasion. This effect was observable with inter-individual heterogeneity in the patient's tumor material. MMP inhibitors effectively reduce glioma invasion, although high concentrations were required in 3-dimensional culture systems. At these concentrations, both compounds revealed a cytostatic, but no cytotoxic effect. Thus, high local concentrations of MMP inhibitors could offer a new therapeutic strategy for the treatment of gliomas.

Animals↗

PubMed1999

Vascularization of human glioma spheroids implanted into rat cortex is conferred by two distinct mechanisms.

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

Animals↗

PubMed1999

Cell-extracellular matrix interaction in glioma invasion.

Astrocytic tumours of the central nervous system express cell adhesion receptors of the integrin superfamily, CD44 and adhesion receptors of the immunoglobulin superfamily. Glioma cells utilize these receptors to adhere to and migrate along components of the extracellular matrix (ECM), which is uniquely distributed and regulated within the brain and the spinal cord. For penetration into healthy brain tissue a number of proteases are expressed, which degrade proteins of the extracellular matrix. Thus, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix and the subsequent destruction of matrix barriers. There is a critical balance between expression of various adhesion receptors and proteases. The tight regulation of critical levels of proteases and receptors expressed by glioma cells or other cells is necessary for the "physiological" behaviour of glioma cells. Shifts in the balance of protein expression determine glioma cell behaviour in their micro-environment and can initiate or influence the complex process of glioma cell invasion. The complex receptor-ECM interaction in glioma cell invasion is discussed focussing upon the role of integrin receptors and matrix-metalloproteinases. Influencing these molecules or their regulation may lead to novel therapeutic approaches in the treatment of malignant glioma.

Animals↗

PubMed1999

Expression of different extracellular matrix components in human brain tumor and melanoma cells in respect to variant culture conditions.

Local tumor invasion into the surrounding brain tissue is a major characteristic of malignant gliomas. These processes critically depend on the interaction of tumor cells with various extracellular matrix (ECM) components. Because only little quantitative information about expression of ECM gene products in general and expression in response to alterations of the surrounding environment is available, the present study was designed. Four human glioblastoma cell lines (U373MG, U138MG, U251MG, GaMG) as well as four human melanoma cell lines (MV3, BLM, 530, IF6) were tested with semiquantitative RT-PCR for their ability to express mRNA of different human ECM components (fibronectin, decorin, tenascin, collagen I, collagen IV, versican). In addition, two human medulloblastoma (MHH-Med 1, MHH-Med 4) and two fibrosarcoma (HT1080, U2OS) cell lines were analyzed. Cells which were grown in DMEM medium containing 10% FCS expressed most of the analyzed protein components. When the same medium, but depleted of ECM proteins by filtrating through a membrane with cut-off at > 100 kD was used, basal mRNA expression of the ECM proteins was changed in most of the examined cell lines. Using serum free conditions, most of the cell lines again showed a variation in the expression pattern of mRNA encoding for the different ECM proteins compared to the other medium conditions. Comparing different cell lines from one tumor entity or different tumor groups, ECM expression was heterogeneous with regard to the different tumor entities as well as within the entities themselves. Migration assays revealed heterogeneous responses between the different cell lines, ECM components and culture conditions, making it difficult to correlate ECM expression patterns and migratory behavior. Our results revealed that all examined cell lines are able to produce ECM proteins in vitro. This suggests that tumor cells can modulate their microenvironment in vitro which has to be taken into consideration for studies related to migration and invasion.

Brain Neoplasms↗

PubMed1998

BCL-2 promotes migration and invasiveness of human glioma cells.

Malignant progression in gliomas is correlated with increased migratory capacity which involves metalloproteolytic activity. Here, we report that ectopic expression of BCL-2 in two malignant glioma sublines markedly promoted glioma cell migration from spheroids and invasion into Matrigel-coated membranes. Invasion of fetal rat-brain aggregates was enhanced by BCL-2. Zymography revealed activation of matrix metalloproteinase-2 (MMP-2) in BCL-2-expressing cells. BCL-2 expressing cells showed an increase in MMP-2/-3/-12 (LN-18), and MMP-9/-12 and cell surface urokinase-type plasminogen activator (u-PA) (LN-229) mRNA and a reduction in tissue inhibitors of metalloproteinases (TIMP)-2 mRNA (LN-229). Taken together, we propose a novel function for BCL-2 in the malignant phenotype of glioma cells, that is, to enhance migration and invasion by altering the expression of a set of metalloproteinases and their inhibitors.

Animals↗

PubMed1998

ECM-mediated glioma cell invasion.

Cell adhesion receptors of the integrin superfamily, CD44, and adhesion receptors of the immunoglobulin superfamily are expressed by high-grade astrocytic tumors of the central nervous system. These receptors are critical for the invasion of these tumors in the nervous system. Glioma cells utilize these receptors to adhere to and migrate along the components of the extracellular matrix, which is uniquely distributed and regulated within the brain and the spinal cord. For this reason, glioma cell invasion into the adjacent brain tissue is dependent on the interaction of glioma cells with the extracellular matrix. The receptor-ECM component interaction is discussed, focusing on the role of cell adhesion molecules of the integrin family and CD44 in glioma cell adhesion and invasion.

Animals↗

PubMed1998

Expression of matrix metalloproteinases in human glioma cell lines in the presence of IL-10.

Matrix metalloproteinases have been implicated to play a vital role in glioma invasion as they degrade extracellular matrix to facilitate the subsequent migration of tumor cells into the surrounding brain tissue. The cytokine Interleukin-10 (IL-10) was detected recently in glial tumors in vivo. Expression of specific IL-10 mRNA as well as blood serum levels of IL-10 in glioma patients increased with malignancy suggesting a functional role of IL-10 in glioma progression. Moreover, glioma cell migration in vitro was enhanced in the presence of IL-10. We therefore investigated the expression of the matrix metalloproteinases (MMPs) stromelysin-1 (MMP-3), 72-kDa collagenase (MMP-2), 92-kDa collagenase (MMP-9), matrilysin (MMP-7) and the human macrophage metalloelastase (MMP-12). In addition, a possible relation between exposure of glioma cells to IL-10 and invasiveness of these cells due to MMP expression was analyzed. Experiments with Matrigel coated Boyden chambers revealed a pronounced dose dependent effect of IL-10 on glioma invasiveness. The synthetic MMP-inhibitor Marimastat markedly reduced cell invasion in the Boyden chambers confirming the significance of MMPs in the process of invasion. Subsequently, the expression level of MMPs and the serine protease uPA was investigated in 7 glioma cell lines (U373, GaMG, U251, GHE, SNB19, U138 and D54) by RT-PCR. In all but one cell line no enhancement of MMP expression by IL-10 was detected. Matrilysin in U373 cells was the only protease found to be upregulated in the presence of IL-10 dependent on cell density. The present data suggest that IL-10 related effects on the invasive properties of the cell lines are not directly mediated by an upregulation of matrix metalloproteinase expression.

Brain Neoplasms↗

PubMed1997

Fast blue, a fluorescent tracer in glioma cell culture, affects cell proliferation and motility.

The azo-dye, Fast Blue (FB), initially employed for retrograde neuronal tracing is increasingly used in cell invasion and migration studies to detect living cells in monolayer and glioma tumor cell spheroid models. As yet, it is assumed that a cell stained with a tracker dye retains the characteristics of the original cell. The following experiments compared the adhesion, migration and proliferation properties of the cell lines U373 and GaMG with and without FB staining. FB staining reduced cell adhesion (P < 0.01) and proliferative activity (P < 0.01) and also had a significant inhibitory effect on cell migration (P < 0.001). From the results presented it follows that FB staining markedly influences basic cell characteristics.

Amidines↗

PubMed1997

Pleomorphic xanthoastrocytoma: report of six cases with special consideration of diagnostic and therapeutic pitfalls.

BACKGROUND: Pleomorphic xanthoastrocytoma (PXA) is a rare clinicopathologic entity that occurs typically in young patients. Improved neuroradiologic techniques, especially gadolinium-enhanced magnetic resonance imaging (MRI), reveal a characteristic tumor appearance. METHODS: We present six cases of PXA operated on with unusual clinical course, elucidating different clinical implications. RESULTS: Two patients showed subsequent progression into a malignant glioma, one case was a primary anaplastic PXA. The latter case had not previously been reported in the literature. Increased mitotic activity seems to indicate a worse clinical course; whereas focal infiltration of the brain does not necessarily lead to malignant transformation. CONCLUSIONS: Surgical removal is the treatment of choice. A consequent follow-up is mandatory in order to detect a potentially malignant recurrence as early as possible and to select patients who need additional therapy.

Adolescent↗

PubMed1997

Mitoxantrone-induced DNA strand breaks in cell-cultures of malignant human astrocytoma and glioblastoma tumors.

In this study mitoxantrone (Mtx) induced DNA strand breaks were measured with the alkaline elution technique in short term cell cultures derived from human gliomas. Glioblastomas or astrocytomas from 5 patients who underwent intracranial surgery were cultured and incubated i h with different concentrations of Mtx (0, 0.01, 0.1 and 1.0 microgram/ml). The alkaline elution method was modified to measure DNA lesions in human gliomas. Mtx induced DNA strand breaks in a dose dependent manner in all cell cultures tested. There was a linear increase of DNA strand break frequency induced by Mtx between 0.01-1.0 microgram/ml. concerning these in vitro data, Mtx might be potentially useful for the treatment of patients with malignant brain tumors.

Aged↗

PubMed1997

Inverse correlation of cell proliferation and expression of progesterone receptors in tumor spheroids and monolayer cultures of human meningiomas.

OBJECTIVE: The progesterone receptor (PgR) can be detected in 60 to 70% of meningiomas using immunohistochemistry] in situ. Whereas in monolayer tissue cultures the PgR is only rarely expressed, we were able recently to demonstrate the preservation of the PgR in fragment spheroid cultures of meningiomas. The aim of the present study was to evaluate the stability of PgR expression in meningioma spheroids in vitro and the correlation of PgR expression and cell proliferation in spheroids and whether meningioma cells reaggregated to spheroids from monolayer cultures to reexpress the PgR again. METHODS: Tumor fragment spheroids (Weeks 1-6) and cell monolayers (Passages 1 and 3) of 15 PgR-positive meningiomas were investigated by immunohistochemistry for the expression of PgRs and their proliferative activity, as demonstrated by positivity for the proliferation-related antigen Ki-67. To study PgR reexpression in reaggregated spheroids, Northern blots were performed. In addition, a reverse transcriptase-polymerase chain reaction technique was established and evaluated in combination with immunohistochemistry. Growth of meningioma spheroids was quantified in the presence of progesterone and the specific antagonist onapristone. RESULTS: The PgR remained stable in spheroids for 6 weeks in 9 of 13 cases that were able to be evaluated. All tumor fragment spheroids exhibited a proliferation index of 5 to 40% Ki-67-positive cells. Monolayer cell cultures, on the other hand, failed to express PgRs but revealed higher proliferation indices (40-90%) to a significant extent. The detection of PgR messenger ribonucleic acid in reaggregated spheroids by means of reverse transcriptase-polymerase chain reaction correlated to the nuclear expression of PgR in immunohistochemistry. Neither progesterone nor its antagonist onapristone altered spheroid growth in vitro. CONCLUSION: The expression of the PgR in meningiomas is preserved in spheroid cultures with low proliferation indices for at least 6 weeks, whereas monolayer cell cultures with a high proliferative activity lack PgR expression. The inverse pattern of Ki-67-positive cells in the outer regions of the spheroids and PgR-expressing tumor cells in the spheroid centers leads us to the conclusion that proliferating meningioma tumor cells do not express PgRs. This might also explain why tumor cell growth in vitro was neither affected by progesterone nor by onapristone. Monolayer cell cultures can be reaggregated to spheroids, the consequence being a reexpression of PgRs and, therefore, a down-regulation of proliferation.

Antineoplastic Agents↗

PubMed1997

Effect of changes in the CD44 gene on tumour cell invasion in gliomas.

Cell adhesion is a critical factor in the multistep process of tumour invasion. CD44 is one of the cell surface adhesion molecules responsible for interaction with hyaluronic acid, a component of the CNS extracellular matrix. The aim of the present study was to demonstrate whether alterations in the CD44 gene might account for different invasive behaviour. EcoRI restriction analysis by Southern blot hybridization revealed several additional hybridization signals in tissue specimens of two out of 16 patients with glioblastoma, indicating DNA rearrangements or point mutations, respectively, within the region of the CD44 gene. Expression patterns of CD44 isoforms in these two rearranged gliomas and in 28 other patients with malignant gliomas were analysed by RT-PCR. All cases displayed only the splice variant CD44H, which acts as hyaluronic acid receptor in glioma tumour cells. Tumour cell invasion was studied with Boyden chamber assays using hyaluronic acid as ligand and functional CD44H blocking antibody. Invasion of cells derived from those gliomas carrying the rearranged CD44 gene locus was decreased by about 50% compared with gliomas without rearrangement, indicating that the altered hybridization patterns in the two glioma samples influenced CD44H mediated glioma cell invasion through hyaluronic acid in vitro. Our results on CD44 isoform expression suggest that, in contrast to other solid tumours, gliomas seem to express only the CD44 variant. Genetic alterations within the CD44 gene might alter the binding domain of the receptor and thus account for different invasive behaviour in glioblastomas.

Alternative Splicing↗

PubMed1996

Progesterone receptors in tumor fragment spheroids of human meningiomas.

Progesterone receptors (PgR) are detectable in about 60-70% of tissue specimens of human meningiomas. Despite these data, PgR are hardly to be found in monolayer tissue culture of meningiomas. Aim of this study was to elucidate whether PgR might be preserved in tumor fragment spheroids of meningiomas maintained in organ culture since the morphological appearance of the original tumor is preserved by this culture technique. Aliquots of meningioma specimens of 25 patients (17 females) were snap frozen in liquid nitrogen immediately after removal. Additionally, monolayer tissue cultures of the same specimen were obtained as primary culture and passage #3. Tumor fragment spheroids were kept on medium-agar with liquid medium overlay and harvested after 1 and 3 weeks in culture. PgR were detected by immunohistochemistry using a rat monoclonal antibody. 18/25 meningioma tissue specimens were positive for PgR. In 8 out of 15 PgR-positive tumors which formed spheroids we could detect PgR in fragment spheroids after 1 and 3 weeks in culture. In contrast, none of the monolayers depicted PgR. PgR is preserved in a considerable amount of tumor fragment spheroids of PgR-positive meningiomas. They remain detectable after 3 weeks of culture whereas monolayer tissue cultures are PgR-negative. Thus, tumor fragment spheroids seem to be a suitable tool to investigate progesterone/antiprogesterone effects in vitro.

Adult↗

PubMed1996

Molecular genetic alterations in pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is a low-grade glioma that may recur as a malignant diffuse astrocytoma such as glioblastoma (GBM). While the molecular genetic basis of diffuse astrocytomas has been studied extensively, PXAs have not been analyzed in detail. We, therefore analyzed DNA from archival primary and recurrent PXAs from eight patients (three grade II PXAs without recurrence, one grade II PXA with recurrence as grade II PXA, two grade II PXAs with progression to GBM, and two grade III anaplastic PXAs with recurrence as grade III anaplastic PXA or GBM) for genetic changes associated with diffuse astrocytomas. Single-strand conformation polymorphism analysis of p53 exons 5-8 revealed migration shifts in two cases, one primary PXA without recurrence and one recurrent grade II PXA in which the primary tumor did not show a shift. DNA sequencing showed two missense mutations in codons 220 (exon 6) and 292 (exon 8), respectively, mutations which have not been previously noted in astrocytomas. Differential polymerase chain reaction analysis demonstrated epidermal growth factor receptor gene amplification in only one tumor, a GBM without allelic loss of chromosome 10 that was the second GBM recurrence of an initial grade II PXA. Loss of heterozygosity studies on tumors from five patients, using three microsatellite polymorphisms on chromosome 10q and three on chromosome 19q, did not disclose allelic loss in any recurrent tumor. These findings suggest that the genetic events that underlie PXA formation and progression may differ significantly from those involved in diffuse astrocytoma tumorigenesis.

Adolescent↗

PubMed1996

Primary Ki-1-positive T-cell lymphoma of the brain--an aggressive subtype of lymphoma: case report and review of the literature.

BACKGROUND: By detection of the Ki-1 antigen, Stein (1985) defined a new entity of anaplastic large cell lymphoma [24]. Apart from our case, only four further cases of Ki-1 positive primary central nervous system lymphoma (PCNSL) have been reported in the literature to date. CASE REPORT: A 63-year-old man presented with two frontal and parietal mass lesions and one ring lesion on computed tomography scan. Clinically, no evidence of brain metastases or abscesses could be found. Immunohistochemical investigations of biopsy specimens revealed a large cell anaplastic T-cell lymphoma positive to Ki-1 antigen. In spite of all therapeutic efforts, the patient died less than 3 months after the onset of symptoms. DISCUSSION: In all cases the clinical course was very rapid, suggesting that Ki-1 positive PCNSL might form an aggressive subtype of lymphomas. Since the radiologic appearance was atypical and clinical diagnosis was not possible, diagnostic biopsy for immunohistochemical diagnosis should be performed.

Brain Neoplasms↗

PubMed1996

Median corpectomy in cervical spondylotic multisegmental stenosis.

Cervical median corpectomy as an alternative to laminoplasty and laminectomy has been suggested as an effective treatment for cervical spondylotic myelopathy (CSM) in cases of multisegmental spondylotic stenosis. We report on our experience with this procedure with particular reference to neurological outcome and complications. Median corpectomy was performed in 17 cases (3 female, 14 male; mean age 59 yrs, (41-80 yrs.) with cervical myelopathy (CM) and radiologically diagnosed multisegmental spondylotic stenosis and spinal cord compression seen on MRI. The degree of stenosis was determined by means of the modified Pavlov's index (ratio between spinal canal width at the level of the intervertebral disc and the diameter of the vertebral body itself). 3/17 patients suffered from acute, 4/17 from subacute and 10/17 from chronic CM. Single level corpectomy was performed in 9 cases, one and a half vertebrae were removed in 2 cases and dual level corpectomy was performed in the remaining 6 cases. All patients received an autologous bone graft and AO - anterior plate stabilization or were stabilized as described by Morscher. Postoperative follow - up was possible in 16/17 cases over a mean time of 13.5 months. Myelopathy was graded according to Nurick's scale. Postoperatively, 12% with chronic CM improved by two grades, 38% (2 pts, with acute, 3 with subacute and 1 with chronic CM) improved by one grade. The other patients remained stable, none showed worsening of their myelopathy. Paresis improved in 92%, sensory deficits in 69%, spasticity in 73%, pain in 60%, and vegetative disturbances in 100% of all patients presenting these preoperative symptoms respectively. One patient died due to esophageal perforation and subsequent lethal mediastinitis caused by screw loosening 4 months following surgery and after initial neurological improvement. 4 other patients experienced screw loosening, three with acataposis, one remained clinically asymptomatic with concomitant graft displacement in two of these. One patient had to be re-operated due to a hematoma at the iliac crest and 2 suffered from a pelvic fracture of the spina iliaca at the site of graft removal. With respect to the neurological improvement, especially to the motor function and spasticity, median corpectomy can be regarded as an effective procedure in selected cases with cervical myelopathy, even when treatment related complications are taken into consideration.

Adult↗

PubMed1995

Quantitative NMR microscopy of multicellular tumor spheroids and confrontation cultures.

In cancer research, tumor spheroids are a well established system to study tumor metabolism resembling the situation in vivo more closely cell monolayers. Spherical aggregates of malignant melanoma cells (MV3) and their invasion into rat brain aggregates have been investigated by quantitative NMR microscopy. Relaxation times (T1, T2) and diffusion parameter images were acquired with an in-plane resolution of 14 x 14 microns2. The authors were able to demonstrate that the morphology of the spheroids can be visualized on these NMR maps. The contrast was mainly manifested in relaxation maps, where average relaxation times T1 = 1.94 +/- 0.17 s and T2 = 42.8 +/- 6.3 ms were obtained for proliferating cells, and T1 = 2.49 +/- 0.31 s and T2 = 104.3 +/- 29.4 ms for the necrobiotic center. The mean diffusion coefficients were 0.59 +/- 0.12 micron2/ms and 0.85 +/- 0.14 micron2/ms, respectively. The authors could follow the dynamic process of tumor cell invasion in the investigated co-culture system. Knowledge about tumor cell migration and tumor cell invasion is essential for the understanding of cancer and its therapy. Quantitative NMR microscopy can study this dynamic process noninvasively and therefore may help to assess the influence of therapy on the micromilieu of these spheroids.

Animals↗