Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.

Biological activity of mutants of human tumour necrosis factor-alpha.

Point mutations in different regions of the tumour necrosis factor-alpha (TNF-alpha) molecule influence anti-tumour cytotoxic/cytostatic activities as well as haemorrhagic tumour necrosis, tumour regression and lethal toxicity in mice. Mutations in the C-terminal region in positions 150 and 155 markedly decrease cytotoxicity for murine L929 fibroblasts and human MCF7 mammary carcinoma cells. Competitive binding experiments with 125I-labelled TNF-alpha revealed that the loss of cytotoxicity is caused by a loss of target cell binding. In contrast to the reduced activity against L929 and MCF7 cells, neither binding to nor cytostatic activity against the human myeloid leukaemia cell lines HL60 and U937 are affected. This target cell type-dependent behaviour is probably due to the fact that L929 and MCF7 cells express different types of TNF receptor compared with myeloid leukaemia cells. While a mutation in position 127 decreases the overall activity of TNF-alpha, a deletion of four N-terminal amino acids does not reduce biological activity. In vivo the TNF mutants differed in their anti-tumour effects and lethal toxicity, but a segregation of anti-tumour activity and toxicity was not observed.

Influence of intensive exercise on insulin-like growth factor I, thyroid and steroid hormones in female gymnasts.

Long lasting intensive physical exercise leads to growth retardation. Short-limbed girls are selected for the training as gymnasts. In a preliminary study with 9 gymnasts a significant decrease of the IGF-I concentration was found after intensive 3-day exercise. This experiment was repeated with 16 girls (11.7 +/- 0.8 years old). The higher the initial DHEA-S and E2 concentration of the gymnasts, the higher were the IGF-I basal levels. The intensive training resulted in the following changes (basal after exercise): IGF-I: 247 +/- 86-->188 +/- 77 ng/ml, T3: 2.4 +/- 0.4-->2.1 +/- 0.3 nmol/l, T4: 96 +/- 15-->98 +/- 19 nmol/l, DHEA-S: 930 +/- 636-->1018 +/- 701 nmol/l, testosterone: 1.5 +/- 0.3-->1.9 +/- 0.4 nmol/l, cortisol: 824 +/- 272-->799 +/- 219 nmol/l. During the 3-day intensive training, the parallel decrease of IGF-I and T3 concentrations in each sportswomen is particularly impressive. Apart from the sequelae of 'negative' selection, the low T3-syndrome, the anti-insulin effect of high GH secretion and the elevated cortisol concentration are responsible for the growth depression, retardation in bone age and the higher incidence of skeletal problems in these gymnasts with 'exercise-induced' delay in development.

[Concentrations of prolactin, LH, estradiol-17 beta and progesterone in swine after biotechnical stimulation with PMSG, HCG and suidor]. / Konzentrationen von Prolaktin, LH, Estradiol-17 beta und Progesteron beim Schwein nach biotechnischer Stimulation mit PMSG, HCG und Suidor.

Concentrations of estradiol-17 beta, progesterone, luteinising hormone (LH), and prolactin were recorded from 9 gilts, following cycle blocking by means of Suisynchron(R)-Prämix and application of PMSG, HCG, and Suidor(R). A radio-immuno-assay which for its quality criteria enabled safe determination of the hormone in peripheral blood had been worked out specifically for prolactin appraisal. HCG application led to blockage of pre-ovulatory LH release in 7 of 9 animals. Possible causes are discussed. Prolactin concentrations during oestrus were differentiated and, for example, were characterised by strong oscillatory variations. With the experimental arrangement used, the boar pheromone Suidor(R) was not found to have any impact upon hormone profiles.

Circannual oscillations of function compared with morphometric changes in the thyroid gland of the Wistar-rat.

To investigate biological rhythms of the thyroid gland circannual oscillations of thyroxine (T4), triiodothyronine (T3), and thyrotropin (TSH) were compared in serum samples of untreated young male Wistar-rats with the circannual changes of thyroid weights and with the relative proportion of colloid, epithelium, and interstitium of the thyroids. Animals were kept under standard environmental conditions, however, lighting conditions simulated the natural day-night changes. Thyroid weights, T4, T3, and TSH showed a statistically significant circannual rhythm with maxima in winter and spring and minima in summer and autumn. The same circannual patterns were observed in the proportion of epithelium and interstitium of the thyroids, while the colloid exhibited an inverse circannual pattern. These data were verified by biomathematical methods, like locally adjusted functional approximation, analysis of variance, and Spearman rank correlation. Our results represent an example for the concordance between functional and morphometrical changes in the course of circannual oscillations. Furthermore, these data confirm our earlier results describing higher T4-levels in the winter time (short-day) and lower serum titers in the summer time (long-day).

Effect of endurance exercise on somatomedin-C/insulin-like growth factor I concentration in male and female runners.

By means of 3 endurance exercises, the effect of a several-hour intensive somatic stress on the changes of the Sm-C/IGF-I concentration was tested during, immediately after and on the day following the exercise. Exp. 1: Marathon with 17 male sportsmen in 2 groups with different glucose supply. Exp. 2: 45-km crosscountry run with 41 males. Exp. 3: Three 20-km runs with 8 young females at intervals of 3 months. In the marathon, no significant changes of the Sm-C/IGF-I concentration were found between the start, half distance and final values. The exogenous glucose supply (continuous or discontinuous) had also no effect. The tendency of a slight decrease of the Sm-C/IGF-I concentration by 0.14 U/ml (p greater than 0.05) was observed between start and finish in the 45 km crosscountry run lasting one hour longer. In the three 20-km runs, reproducible, slightly increased levels were measured at the end, whereas a decrease to the initial value or even below was detected on the following day (p greater than 0.05). The insignificant alterations of the Sm-C/IGF-I concentration measured in the 3 variants of races show that neither the hormonal changes stimulating the Sm-C/IGF-I synthesis (e.g. increase of GH and prolactin) nor inhibiting factors (energy deficiency) clearly dominate during strenuous exercises. The binding of carrier protein prevents great variations of the Sm-C/IGF-I level even under the condition of 3- to 4-hour extreme endurance exercises.

Growth of mammary epithelial cells in breast-cancer biopsies correlates with EGF binding.

In order to understand the role of EGF in the development of human mammary epithelial tissue, we analysed the binding of 125I-EGF to sections of breast cancer biopsies. A mean specific 125I-EGF binding of 8.9 fmol per mg protein was estimated in thin sections of 17 breast cancer biopsies. Microautoradiographic analysis of 125I-EGF binding to the tissue sections was applied to demonstrate that EGF was bound predominantly to mammary epithelial cells. The binding was clearly correlated to the number of mitoses of mammary epithelial cells in the same samples. The highest EGF binding and proliferation rates were found in biopsies from breast cancer with axillary lymph-node metastases.

[Tumor marker diagnosis in non-seminomatous testicular tumors using human chorionic gonadotropin (HCG) and alpha fetoprotein (AFP)]. / Tumormarkerdiagnostik bei nicht-seminomatösen Hodentumoren mittels Humanchoriongonadotropin (HCG) und Alpha-Fetoprotein (AFP).

In 28 patients with non-seminomatous testicle tumour the tumour markers human chorionic gonadotropin (HCG) and alpha-fetoprotein (AFP) were determined radioimmunologically and enzymeimmunologically, respectively. While tumours with chorionic carcinoma (n = 8) always were marker-positive, in the embryonic carcinoma in 2 out of 10 cases falsely negative findings appeared. On 5 patients the biochemical monitoring of the course of the testicle tumour disease is demonstrated in detail by means of HCG and AFP and estimated as very helpful method. Advantages and problems of the marker diagnostics are shown and discussed. The positive marker findings were absolutely evident for a metastasation. On the other hand, marker negativation was not always to be equated with absence of a tumour and demanded a further control of the patient by means of all other available methods of diagnostics up to the second-look-operation.

The use of glass as solid phase in enzyme immunoassay as exemplified by the detection of circulating immune complexes.

This article describes a solid-phase enzyme immunoassay for the quantitative determination of circulating immune complexes based on the covalent binding of proteins to glass. A quantitative comparison between adsorption to polystyrene and covalent binding to glass gave a protein higher concentration on the glass support. This leads, in connection with the strong attachment of the protein, to a higher sensitivity and precision in immunoassay. The binding of C1q to glass as solid phase offers the possibility to reuse the protein as well as the glass support after accomplishment of the immunoassay which can reduce the cost of the assay in routine work dramatically. Furthermore, the immobilization of C1q to glass gives an extreme stability of the protein which means that the c1q-coated glass supports can be used up to one year under routine conditions.

Effect of dextran on the release of gonadotropin-releasing hormone (GnRH) injected into rats: plasma GnRH and gonadotropin response.

Prolonged release of the peptide gonadotropin-releasing hormone (GnRH) from its aqueous solution was achieved by addition of the polymer dextran (Mw ∼ 500,000). This effect observed in an in vitro system was caused by a decrease of the diffusion coefficient of the peptide. When GnRH was intramuscularly injected into male rats, the addition of dextran to the injected peptide solution led to a prolongation of the GnRH plasma level at the expense of its peak value. This change can be explained by a decrease of the absorption rate of GnRH into blood, which parallels the in vitro observation. As a result, the gonadotropin response to GnRH was stronly increased.

Purification of a growth inhibitor for Ehrlich ascites mammary carcinoma cells from bovine mammary gland.

A growth inhibitor for Ehrlich ascites mammary carcinoma cells in vitro has been purified from bovine mammary gland. The purification procedure involving homogenization and differential centrifugation under hypotonic conditions, ammonium sulfate precipitation, ultrafiltration, gel chromatography and preparative polyacrylamide gel electrophoresis (PAGE) yielded an inhibitor showing half-maximal inhibition of cell proliferation in concentrations of 1-3 ng protein per ml. Upon 125I labelling and analysis by SDS gel electrophoresis, most purified preparations revealed a single band of 12-14 kD, likely to be representative for the inhibitory protein. The inhibitor was shown to affect resumption of proliferation of stationary cells; however, it was inactive towards cells stimulated by incubation with medium before adding the inhibitor. The inhibitor is heat-labile, does not act by exhausting essential components of culture medium, and its action is antagonized by insulin.

[Coagulation of 125I bovine fibrinogen]. / Untersuchungen zur Gerinnbarkeit von 125I-Rinderfibrinogen.

Bovine fibrinogen was labelled with 125I using the chloramine T method or the iodogen method and the clottability of the preparations was studied in vitro and in vivo. Three days after radioiodination the in vitro clottability was 82.3% (chloramine T) and 80.4% (iodogen), respectively. When the solutions were allowed to stand at 4 degrees C for 13 days, the in vitro clottability decreased to 70% or 60%, respectively; either preparation was practically unclottable after 25 days. The preparations were administered to rats three days following radioiodination. They showed the same elimination rate and, on thrombin infusion, the same clottability. 125I-labelled (chloramine T) bovine fibrinogen stored in solution at -20 degrees C for 38 days showed a clottability of 76%, the in vivo clottability was unchanged.

Proteolytic activities associated with plasma membrane preparations from tumour cells in enzootic bovine leukosis and from normal bovine lymphoid cells.

Proteolytic activities associated with plasma membrane preparations from cells of tumourous lymph nodes in enzootic bovine leukosis and from normal bovine lymphoid cells have been studied. 125I-Casein-Sepharose has been used as a substrate. No significant difference between both cell types has been found in total proteolytic activity at different pH-values as well as in the number and type of proteinases, the membranes seem to be equipped with. The main proteolytic activities present in both types of membranes are: One (or two) pepstatin-inhibitable aspartate-proteinase(s) with maximum activity at pH 3 and 5, a leupeptin-inhibitable, presumably cysteine-proteinase, maximally active at pH 4, very small activity(ies?) between pH 7 and 8 of uncertain nature, and a plasminogen-activator with maximum activity around pH 8. Autodigestion of plasma membranes from normal lymph node lymphocytes has been performed choosing conditions optimal for the different proteinases. The only specific proteolysis observed is the splitting of a glycoprotein (151.5 kD), which is present in the plasma membranes of normal lymph node lymphocytes but not in the plasma membranes of peripheral blood lymphocytes and tumour cells, under conditions optimal for the aspartate-proteinase. A possible role of this specific proteolysis is suggested for tumouri-genesis and/or loss of sessility of normal lymphocytes.

[Radioimmunologic technics for luteinizing hormone determination in cattle and sheep]. / Radioimmunologische Testmethoden zur Luteinisierungshormonbestimmung bei Rind und Schaf.

The antigen relationship which exists between luteinising hormone of various animal species, on the one hand, and human luteinising hormone as well as human chorionic gonadotrophin, on the other, was the background against which possible approaches were tested to the preparation of a heterologous in-vitro test for serum LH determination in cattle and sheep. The following conclusions may be drawn from the results so far obtained: --A homologous or heterologous test with specific relevance to the determination of human LH, on the basis of hLH-anti-hLH or hLH-anti-hCG is not suitable for the determination of LH in animal serum (cattle, sheep). --Quantitative and specific measurement of LH concentrations in the blood of cattle (sheep) is possible by means of an ovine-LH-anti-ovine/bovine-LH (or anti-bovine-LH) test system which is based on cross-reactivity between luteinising hormones of cattle and sheep. --Differentiated measurement of various LH concentrations in animal serum will be possible under certain specific assay conditions, with ovine LH as tracer, ovine or bovine LH as standard, and hCG antiserum. Yet, the clinical value of that heterologous radio-immuno-assay method is attributable primarily to its potential to measure peak values within the cycle to determine the time of ovulation rather than to the built-in possibility to determine base values of the cycle.