A comparative quantitative analysis of laminin-5 in the basement membrane of normal, hyperplastic, and malignant oral mucosa by confocal immunofluorescence imaging.

Laminin-5 (Ln-5) is a heterotrimeric basement membrane (BM) molecule (alpha3beta3gamma2). It is a principal protein constituent of the anchoring filaments, which connect the BM with the hemidesmosomes of the basal keratinocytes and possess a crucial function in keratinocyte adhesion. Confocal immunofluorescence imaging is introduced for a quantitative evaluation of the Ln-5 content in the BM of oral squamous epithelium. The BM of normal oral mucosa was used as a reference (100%) for comparative analysis and showed a nearly uniform Ln-5 immunofluorescence intensity (99-100%). In all hyperplastic lesions of oral mucosa, the Ln-5 immunofluorescence intensity was increased (107-141%). The increased Ln-5 content in the BM of hyperplastic lesions suggests an increased keratinocyte-BM adhesion, possibly resulting in a higher stability of the oral mucosa. In contrast, in the oral squamous cell carcinoma (OSCC) invasive front, the remaining BM segments were characterized by a decrease in Ln-5 immunofluorescence intensity (35-74%). A stronger decrease of Ln-5-linked kerationocyte-BM adhesion correlates with a higher tumor grade. Because in central areas of carcinoma BM segments with a normal Ln-5 content could be demonstrated, the fundamental Ln-5 diminution in BM segments of the invasive front should be considered as an invasion-associated phenomenon.

Evaluation of microvessel density by computerised image analysis in human renal cell carcinoma. Correlation to pT category, nuclear grade, proliferative activity and occurrence of metastasis.

A high microvessel density is suspected to favour tumour progression and the occurrence of metastasis. To elucidate the significance of abundant vessels for the behaviour of human renal carcinomas, the microvessel density of 110 renal cell carcinomas was correlated to pT category, nuclear grade, proliferative activity, occurrence of metastasis and relapse-free survival interval. The microvessels were quantified using CD31 immunostaining of endothelial cells and computer-aided image analysis. The rules for reproducible microvessel counting, as defined by Weidner, were strictly observed. A statistically significant relationship between the microvessel density and nuclear grade, proliferative activity, occurrence of metastasis and relapse-free survival was found; only for tumour size could no such relation be seen. Perplexingly, there is a diminution of microvessel density in association with increasing nuclear grade, proliferative activity, relapse-free survival interval and frequency of metastasis. This finding is contradictory to the hypothesis that an increasing microvessel density indicates a worsening prognosis.

The association between tumour progression and vascularity in myxofibrosarcoma and myxoid/round cell liposarcoma.

Angiogenesis is an important factor in the morphological progression and metastasis of many solid tumours. We studied two homogeneous series of myxofibrosarcoma (MFS) and myxoid/round liposarcoma (MRLS), characterised by distinct vascular patterns and correlated the intratumoral microvessel density (IMD) with morphologic progression in both types of sarcoma. In our study, 43 cases of MFS and 42 cases of MRLS were graded according to established diagnostic criteria. For evaluation of IMD, representative sections were stained immunohistochemically for CD31. After selection of "neovascular hot spots", IMD was calculated by measuring the endothelial surface within twenty 200x fields in relation to the total analysed area. In addition to the correlation of IMD with histological grades of malignancy, a correlation of IMD with the inflammatory infiltrate in MFS was done. To determine whether vascular endothelial growth factor (VEGF) and its receptors, KDR and flt-1, may play a role in the progression of both types of sarcomas, we used mRNA in situ hybridisation (ISH) to study VEGF, KDR and flt-1 expression in selected cases. In addition, the expression of thrombospondin-1, which has been reported to inhibit angiogenesis, and of collagen type I was studied using mRNA ISH. Cases of MFS varied histologically from hypocellular, mainly myxoid, neoplasms (low-grade malignant, 18 cases) to intermediate-grade malignant lesions with increased cellularity and mitotic activity (13 cases), and high-grade malignant cases with marked pleomorphism, high proliferative activity and areas of necrosis in many cases (12 cases). Cases of purely low-grade myxoid liposarcoma (16 cases) were characterised by low-cellularity, mucin pooling and plexiform vasculature. In combined MRLS, these hypocellular areas were admixed with hypercellular, round cell areas (5-80% of the analysed tumour area; 23 cases), and in round cell liposarcoma (three cases) rounded tumour cells predominated (>80% of the analysed tumour area). The average IMD in intermediate and high-grade malignant MFS (4.03 and 4.09, respectively) was significantly higher than in low-grade malignant MFS (2.73). Correlation of vascularity with the inflammatory infiltrate in MFS showed increased IMD only in cases with abundant neutrophils; most of these cases were high-grade malignant neoplasms. In contrast, no statistical correlation between morphological progression and IMD was seen in myxoid liposarcoma (6.08), MRLS (6.57) and round cell liposarcoma (4.07). VEGF mRNA was expressed by tumour cells in all histological grades of MFS and MRLS. VEGF receptor mRNA was weakly expressed by endothelia of newly formed blood vessels in both entities. Interestingly, tumour cells of all analysed cases of MFS strongly expressed collagen type I and thrombospondin-1, while these proteins were not detected in tumour cells of MRLS. In conclusion, morphologic tumour progression in MFS is associated with increased IMD, whereas, in MRLS, no such correlation is seen. Whereas VEGF and VEGF receptor mRNA were expressed in both entities, a characteristic expression profile of collagen type I and thrombospondin-1 in MFS emerged. Further studies are necessary to correlate vascularity and clinical course in MFS and MRLS.

Radioimmunodetection of neuroblastoma cells with I-131-radiolabelled antibodies.

The murine anti-neuroblastoma monoclonal antibodies 15/7 and 19/1/4 should be tested for specific radiolocalization of neuroblastoma by immunoscintigraphic imaging of this tumour growing in mice. Radioiodination of both antibodies was done by chloramine-T method resulted in an immunoreactivity of 75%. The calculated specific activity varied from 51.1 to 126.2 kBq/microgram IgG. In each case, about 500 kBq of labeled antibodies were intraperitoneally injected into human neuroblastoma (SK-N-MC, SK-PN-DW and IMR 5) xenografted severe complete immunodeficient (SCID) mice. Whole-body scintigraphy was performed daily by a scintiscanner to localize the tumour site. After last scanning principal organs were removed and their I-131-uptake was determined by measuring the impulse rate. The best scintigrams were done with I-131-19/1/4 at the second day after antibody injection. Radioconjugates were accumulated at highest in the tumour at the third day after application of 15/7 and 19/1/4 with a tumour uptake of 0.4 and 2.2 per cent of injected dose per gram (%ID/g), respectively. The 15/7-moAbs was accumulated approximately 9-fold higher in the SK-N-MC and SK-PN-DW grafts than in principal organs, whereas the tumour/non-tumour-ratio of the 19/1/4 moAb was 3:1. The results indicate the efficacy of these two neuroblastoma antibodies in radiolabelling and their usefulness for tumour imaging of neuroblastoma engrafted SCID-mice.

[Effect of the photometric resolution on the precision of microphotometric measurements]. / Untersuchungen zum Einfluss der photometrischen Auflösung auf die Genauigkeit mikrophotometrischer Messungen.

From investigations on microautoradiographs of suitable biological structures, it is concluded, that for the determination of photometric quantities without loss of accuracy a photometric resolution of n = 10 intervals of optical density is sufficient. A low photometric resolution allows to represent the local distribution of substances more clear and the expense of analysis is reduced.

[Quantitative histochemical study of pancreatic islets of sand rats (Psammomys obesus) in diabetes mellitus I. Cytophotometric measurement of insulin content]. / Quantitativ-histochemische Untersuchungen an Langerhansschen Inseln von Sandratten (Psammomys obesus) während der Ausbildung eines Diabetes mellitus. I. Cytophotometrische Messung des Insulingehalts.

The insulin-content in islets of Langerhans on sand rats with disturbances of the carbon hydrate tolerance (reference-, impaired glucose tolerance-, and diabetic group) is measured cytophotometrically. In the IGT-group (impaired glucose tolerance) is a decrease of the insulin content in B-cells detectable. However in the diabetic group of sand rats is the insulin-content increased. This results demonstrate the stimulation of the B-cell function in the islets of Langerhans. The diabetes mellitus of sand rats is characterized by disturbed glucose tolerance hyperinsulinaemia, and elevated readiness to insulin secretion. That are characteristics of the human type-II-diabetes (insulin-independent). In conclusion, the sand rat is a biological model of the human type-II-diabetes (NIDDM).

Experimental studies on the stem cell concept of liver regeneration. I.

There are increasing references for the existence of a hepatic stem or progenitor cell system as well as its participation in the physiological as well as reparative regeneration of the liver and in carcinogenesis. For the physiological regeneration the existence of a dynamic "cell-renewal" system finds increasing consideration and in the "streaming liver concept" (Zajicek et al. 1985) its functional expression. This concept is still under discussion. The present paper tries to check this animal-experimentally (Wistar rats) under use of two different thymidine analogues (3H-thymidine and Bromodeoxyuridine). In different time intervals after labelling (1 h, 14, 30, 60, 90, 120 d) a shift of the labelling bias or a migration of the hepatocytes in the liver acinus (Rapaport) in portovenous direction could be shown. The average migration speed is 0.575 microm or 0.0315 cell positions per day, the cell production rate is one in 31.5 days. The present paper results support the inclusion of a stem or progenitor cell system into the physiological regeneration of the liver and allow the classification into the "streaming liver concept" (Zajicek et al. 1985).

Experimental studies on the stem cell concept of liver regeneration. II.

There is increasing evidence in support of the existence of a hepatic stem or progenitor cell system as well as its participation in the physiological as well as reparative regeneration of the liver and in carcinogenesis. In the present paper it will be demonstrated that under the condition of a simultaneous intoxication with allyl alcohol and tetrachlorcarbon a regenerative process occurs, which is composed of two distinct partial processes: 1. a "proliferative wave" of the persisting, non-necrotic differentiated hepatocytes of the acinus, starting at 24 h after intoxication and ending after >48 h, and 2. a proliferation of cells at the rim of the portal fields, which afterwards enter the acinus and differentiate into hepatocytes, starting at 48 h after intoxication. These results support the participation of a stem or progenitor cell system in the reparative regeneration of the liver.

On methods of silver grain counting in histoautoradiograms.

Visual silver grain counting in histoautoradiograms is a slow and tedious procedure. In this study different methods of automation of grain counting, possibilities of use as well as their advantages and disadvantages are critically compared. By means of autoradiograms of 3H-phenylalanine-labelled rat liver specimens the possibilities of automated image analyses are demonstrated. The use of these techniques enables rapid counting of silver grain densities over cell nuclei and cytoplasm.

[Proliferative activity of mesenchyme and epithelium in teratogen- affected palatogenesis]. / Die proliferative Aktivität von Mesenchym und Epithel in der teratogen beeinflussten Palatogenese.

In mesenchymal cells of the palatal shelves there are two maxima with a 3H-thymidine labelling index of more than 30%. In the teratogenically altered palatogenesis there is only one peak in the labelling index one day before horizontalization of the palatal shelves. In the proliferative rate of epithelial cells are no principal differences between normogenesis and teratogen altered palatogenesis.

Interference microscopic determination of the section thickness of different paraffin-embedded organ tissues.

With respect to the significance of the section thickness in quantitative-morphological studies the thickness of different paraffin-embedded tissue sections was determined by interference microscopy. Within the range of 3-5 microns the measurements revealed accordance of the measured section thickness with that adjusted on the sliding microtome. In the range surpassing 5 microns the section thickness obtained was in all cases lower than that adjusted on the microtome. The deviations were up to 40%.

[Application possibilities of an automatic image analyzer ("Quantimet 720") in bone histomorphometry]. / Untersuchungen zur Einsatzfähigkeit eines automatischen Bildanalysegerätes ("Quantimet 720") in der Knochenhistomorphometrie.

By the way of proofing of the preventive estrogen effect on the ovariectomy osteoporosis in rats the authors collected experiences in measuring of two histomorphometric parameters "trabecular bone volume" and "trabecular bone surface" with the automatic image analyser Quantimet 720. This method demands thin bone polishes or cuts, a very rich in contrast and selective colouring. The measurement with this device spares time and gives reliable results for "trabecular bone volume", but less reliable values for "trabecular bone surface".

The budgerigar (Melopsittacus undulatus) as endocrinological test model. Thyroxine transport in blood.

By comparing in-vivo and in-vitro investigations the transport protein of thyroxine in the budgerigar (Melopsittacus undulatus) was determined. Electrophoretic and auto-radiographic methods were applied. Both investigations show the alpha-globulin to be a thyroxine-binding protein. The contradictions to other investigators are discussed. The experimental animal as an endocrinological model of the thyroid for questions of human medicine is recommended.

Killing of untreated target cells by nonimmunized rat macrophages in vitro. I. Studies on the type of effector cells and their capacity to destroy allogeneic target cells.

Spleen cells derived from unsensitized Wistar rats lysed untreated, allogeneic liver cells of high passages (93rd until 159th subculture) as well as allogeneic tumor cells in mixed cultures. On the other hand, rat liver cells between passages 42 and 56 and allogeneic or xenogeneic fibroblasts were not destroyed by rat spleen cells in vitro. The degree of target cell lysis was dependent on the incubation time and effector-target cell ratio. In the rat spleen, the macrophages were detected as effector cells. Rat peritoneal cells showed the same effects as rat spleen cells. Trypsin treatment caused no loss of cytotoxicity of rat macrophages. Thymocytes and blood lymphocytes of rats were not cytotoxic against allogeneic or xenogeneic target cells. The macrophages apparently destroyed target cells by a nonphagocytic form of cell contact. Unstimulated spleen cells of guinea pigs, Syrian hamsters, CBA mice and rabbits were not capable to destroy target cells in allogeneic and xenogeneic systems.

[Proliferation kinetic examinations on an experimental renal tumour with high portion of hypoxic tumour cells (author's transl)]. / Proliferationskinetische Untersuchungen an einem experimentellen Tiertumor mit hohem Anteil hypoxischer Tumorzellen.

By autoradiographic examination by means of pulse labelling with 3H-thymidine cell cycle times were determined in solid Ehrlich carcinomas (early carcinomas) in order to obtain comparative values for irradiated tumours. The subcells of the initially labelled cells near the capillaries were found to distribute themselves in the course of 48 h over the whole tumour and to emigrate to hypoxic tumour areas.

The proliferative activity of the adrenal cortex as influenced by carcinogenic and noncarcinogenic substances.

A single oral toxic dose of carcinogens (N-nitrosomorpholine, aflatoxin B1, N--methyl-N-nitrosourea, urethan) causes a significant increase of the mitotic rate in the adrenal cortex 48 hours after application. Both, the control animals (water via stomach tube) and the animals treated with acetylsalicylic acid, arsenic, phenol and actinomycin D do not produce this effect. These findings confirm our previous results obtained by administration of acetylaminofluorene, diethylnitrosamine, dimethylnitrosamine and trinitroso-trimethylen-triamine. It is possible that the increased mitotic activity of the adrenal cortex is correlated with the substances applied.

[Cell proliferation kinetics in the small intestine of rats with injured kidneys (author's transl)]. / Das Verhalten der Zellproliferationskinetik im Dünndarm nierengeschädigter Ratten.

Cell proliferation studies were performed in the duodenum, jejunum and ileum of 10 rats 5 months after 5/6 nephrectomy. The control group consisted of 8 unoperated rats. The results showed no differences in the labeling index, in the localisation of the proliferation within the crypts of Lieberkühn and in the migration of labeled cells from the crypts to the top of the villi in both groups. These results may be explained by the small degree of renal insufficiency.

[Studies of the cell cycle of experimental animal tumors following X-ray treatment]. / Zellzyklusuntersuchungen an experimentellen Tiertumoren nach Röntgenbestrahlung.

With the aim to contribute to optimized radiotherapy the proliferation kinetic parameters in experimental animal tumors were determined. As compared to non-radiated tumors, a single X-ray dose of 3 Gy accelerated the time course of the mitotic cycle which may be interpreted as a repair response. In contrary, following a single irradiation with 12 Gy the mitotic phases were delayed in their temporal course. Surprisingly, fractionated irradiations of these tumors yielded significantly less favorable results (no tumor regression, shorter rates of survival) in comparison with those after a single high X-ray dose. Obviously, this is due to a great share of hypoxic tumor cell areas in solid Ehrlich carcinomas which cannot irreversibly be impaired by low radiation doses.

[Laminin-5 in diseases of the oral cavity]. / Laminin-5 bei Erkrankungen der Mundhöhle.

The basement membrane molecule laminin forms a family of proteins. Laminin-5 was identified as key protein in the anchoring filaments of the basement membrane. The anchoring filaments connect the basement membrane to the epithelial cells and together form the epithelial adhesion complex. Disturbances in the epithelial adhesion complex result in subepithelial blistering dermatosis. Autoantibodies against laminin-5 are found in cicatricial pemphigoid. Mutations in genes of laminin-5 with loss of the protein cause epidermolysis bullosa Herlitz. It can be determined during prenatal genetic diagnostic testing. Laminin-5 supports migration of keratinocytes and forms the basis for migrating keratinocytes in oral wound healing. Positive re-epithelialization via laminin-5 has been the subject of experimental studies. Oral squamous cell carcinomas are found to have a focal loss of laminin-5 in conjunction with a loss of the cellular receptor, and increased synthesis of laminin-5 by budding tumor cells has been observed along with deposition of the protein in the stroma. As a result of these observations laminin-5 has been suggested as a route of invasion. Quantitative assessment of this phenomenon and the modulation of laminin-5-tumor cell interaction offer new and hopeful means of describing and affecting the invasive behaviour of oral squamous carcinoma.