The scientific heritage of Professor Zilber and his contribution to virus-genetic theory of malignant tumours.

Effect of thymectomy on tumor development and on T and B lymphocytes in tumor-bearing rats.

Tumor growth and changes in T and B lymphocyte ratio in spleen, draining lymph node and peripheral blood of thymectomized, irradiated rats, reconstituted with syngeneic bone marrow transplanted at various time intervals with MC-1 fibrosarcoma cells were followed. Control nonthymectomized or "sham" operated rats were transplanted an equal dose of tumor cells. Thymectomy and irradiation reduced the numbers of T lymphocytes in all lymphoid organs, while the enhanced numbers of B cells are probably related to reconstitution with cells of syngeneic bone marrow. The time interval between thymectomy, irradiation and transplantation of tumor cells proved to be a limiting factor for tumor growth and changes in T and B cell ratio. Early transplantation of tumor cells (7 days after irradiation) resulted in an enhanced resistance to tumor development, a reduced tumor growth rate and a progressing decline in the number of T cells. If the interval between thymectomy and tumor cell transplantation lasted 4 weeks, the T cell population became partially regenerated, and tumors grew progressively in correlation with a continuing T lymphocyte depletion. The results are discussed in terms of the role of various T cell subpopulations and the significance of residual, thymectomy- and irradiation-resistant T lymphocyte population, vital for a preservation of T cell immunological functions.

T and B lymphocytes of the regional lymph nodes in patients with carcinoma of uterine cervix.

A comparative study of the peripheral blood and the regional lymph nodes lymphocyte subpopulations at 25 patients with carcinoma of uterine cervix was carried out by non-immune rosettes and immunofluorescence methods. The lymph nodes of ten women not affected with any disease, but operated on the occasion of the traumas were investigated as a control. The patients had a significant reduction of the relative amount of T cells and increase of number of B cells in their lymph nodes as compared with the control group. A significant variability of immune indices of the regional lymph nodes as compared to those of peripheral blood was shown. It was pointed out that the presence of the metastases in the regional lymph nodes is one of the possible reasons of this phenomena. The possible significance of the results is discussed.

Cell-mediated immune reactions directed against syngeneic methylcholanthrene-induced mouse sarcomas: low reproducibility of the results obtained with the tube modification of leukocyte adherence inhibition assay.

The value of the tube modification of leukocyte adherence inhibition (LAI) assay for assessment of cell-mediated reactions directed against tumor-associated antigens of transplantable MC-induced murine sarcomas was examined. It was established that the adherence of leukocytes isolated from spleens of tumor-bearing mice is specifically inhibited with 3 M KCl extracts of corresponding tumor cells. However, the reproducibility of the results obtained with the tube modification of LAI assay was found to be rather low. Using the same experimental protocol and the same 3 M KCl extracts of the sarcoma and control tissues, significant differences between adherence in tumor specific and control samples were only found in approximately 50% of the experiments.

Correlation of extracts obtained by high efficiency gel chromatography of lactic dehydrogenase virus infected mouse serum and cytosol from human tumors using leukocyte adherence inhibition assay.

Immunochemically active fractions were obtained using Separon Hema-300-glc(R) from serum of lactic dehydrogenase virus (LDV) infected mice and from homogenates of human tumors. The mixture of proteins of tumorous origin from the cytosol giving a positive reaction in the leukocyte adherence inhibition (LAI) test was found in the same fractions showing maximum of absorbance at 340 nm in the spectrophotometer and a corresponding peak in the refractometer. Analogous peaks were not proved in material obtained from healthy controls, but they were found in some human placentas and fetal organs. The LDV fraction obtained from mouse serum served as a "control" antigen in LAI test for human tumor testing, and results corresponded with those obtained using cytosol specific for the tumor under study.

Peripheral blood lymphocyte nucleoi of rats with Yoshida ascitic sarcoma and the effect of endotoxin.

Nucleoli of lymphocytes were studied in the peripheral blood of male Wistar rats bearing Yoshida ascitic tumor untreated and treated with Escherichia coli endotoxin. Both the neoplastic process and the endotoxin produced an increase of peripheral lymphocytes with "active" nucleoli in number. The treatment of experimental animals bearing the tumor with the endotoxin decreased their mortality and enhanced the increase of the lymphocytes with "active" nucleoli in their peripheral blood.

Detection of specific immunoreactivity by LAI assay in patients with malignant melanoma of uveal tissue.

To monitor the specific antitumor immunity to patients with malignant melanoma of the uveal tissue, use was made of the 20-hr modification of the leukocyte adherence inhibition (LAI) test. Simultaneously, changes in the proportionally of T (total and active) and B lymphocytes in peripheral blood were followed in the same patients by the E and EAC rosette formation test. Peripheral blood leukocytes from patients with malignant melanoma of the uvea, cultured in vitro for 20 hr with syngeneic and allogeneic tumor extract showed a significantly lower adhering capacity than the same cell population cultured without the specific tumor extract (21/22). A retest 3 months after tumor removal showed the percentage of positive cases to have declined (11/19). Leukocyte adherence of control donors, cultured with and without the tumor extract remained unchanged. A follow-up of immunocompetence in patients with malignant melanoma of the uvea, based on proportionally changes of total T, "active" T and B lymphocytes yielded information of an orienting value only. The results of investigation of the various cases proved to be considerably divergent and failed to present a reliable picture of the patient's immunological status.

T and B lymphocytes in lymph nodes and spleen of patients with Hodgkin's disease.

Lymphocyte surface markers of 100 tissue specimens (67 lymph nodes and 33 spleens) of 40 patients with Hodgkin's disease were studied by means of spontaneous E-rosette formation and direct immunofluorescence method. The occurrence of active and total T cells was signigicantly higher (p less than 0.001) in the tumor-involved lymph nodes and spleens, while the frequency of B cells was practically the same in the histologically negative lymph nodes and spleens. In the tumorous spleen of 6 untreated patients the frequency of active and total T cells was higher in the tumorous tissue specimens than in the surrounding histologically negative tissue specimens. No correlation has been found between the simultaneously investigated peripheral T and B cell counts, clinical stage and the histological type of the tumor.

Tumor dormancy (a review).

Immunological studies in breast cancer lymphoid populations.

Anti-tumor antibodies in lung cancer patients. Immunofluorescence study using various indicator cells.

By means of indirect immunofluorescence a number of primary lung cancer patient sera and control sera were tested for anti-tumor antibody activity on living tumor cells as a substrate. Antibodies against surface antigens were the most frequently detected in autologous system (in 65%) on cells derived from fresh surgical material of lung cancer. They were also found in 50% of cases using tumor cells from primary short-term culture. When established cell line of lung cancer was used (E-14) in allogeneic system, the antibodies were detected in only 22% of examined lung cancer sera. Absorption of positive sera with homogenates of normal tissues did not abolish their specific activity. Positive reactions were confined to squamous cell type of bronchogenic carcinoma.

BCG vaccination in Hodgkin's lymphogranuloma.

Results of active non-specific immunotherapy in 25 patients suffering from Hodgkin's disease are given. All of them, being at the stage of a complete remission (residual disease) were divided into two groups according to the clinical stage: group 1 at the stage I and II and group 2 at the stage III and IV. To each vaccinated patient a non-vaccinated control, being at the same stage of disease and of approximately the same age, was selected by applying the method A--B. Results as to the survival time and the remission duration were in both vaccinated groups significantly longer although less significant in patients with an advanced stage of the disease. The present report is to be considered preliminary and the research work is carried on.

Carcinogenicity and polarographic behaviour of dibenzo[a,h]pyrene, 4,11-diazadibenzo[a,h]pyrene and 7,14-diazadibenzo[a,h]pyrene.

In a simultaneous test of carcinogenicity the writers have studied the activities of dibenzo[a,h]pyrene (I), 4,11-diazadibenzo[a,h]pyrene (II) and 7,14-diazadibenzo[a,h]pyrene (III). These compounds were dispersed in paraffin disks and subcutaneously implanted in rats. Each experimental group consisted of 30 animals. The number of sarcomas induced by (I) and (II) was 23 and 16 respectively. The compound (III) has proved wholly inactive. Tumorigenicity of (I) and (II) was found to be proportional to their electron donation and inversely proportional to their electron acceptance in the performed polarographic test. Inactivity of (III) is being discussed from the aspect of molecular geometry.

Biosynthesis of cholesteryl 14-methylhexadecanoate in the liver of rats bearing transplantable tumors and during chemical carcinogenesis.

Biosynthesis of cholesteryl 14-methylhexadecanoate, cholesteryl palmitate and cholesteryl stearate was studied in the liver of rats bearing the Walker 256 carcinoma. Zajdela hepatoma and during chemical carcinogenesis following the administration of benzo[a]-pyrene. An up to 9-fold enhanced production of all these esters was found in liver homogenate during the 10--16th day after Walker tumor transplantation. Only the enzyme system esterifying cholesterol in the cytosol at pH 6.5 was stimulated while the activity of similar enzymes in mitochondria, microsomes and cytosol at an acid pH were not affected. Activity of the cytosol enzyme esterifying cholesterol at pH 6.5 was also enhanced during the active growth of Zajdela hepatoma and during the period of chemical carcinogenesis characterized by the appearance of first palpable subcutaneous tumors. Enhanced activity of cholesterol esterifying enzymes in the liver exactly coincided with periods of elevated levels of cholesteryl 14-methylhexadecanoate in the liver and blood plasma as described earlier. An increased demand of the tumor-bearing host for this cholesteryl ester utilized as a co-factor for enhanced protein synthesis is obviously met by its stimulated production in the liver tissue.

Morphologic changes in the gastric mucosa of rats after administration of N-methyl-N'-nitro-N-nitrosoguanidine. Electronmicroscopic study.

Electron microscopic findings of the effects of exposure to the cancerogenic substance N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) upon the gastric mucosa of rats are presented. MNNG was administered in drinking water at a concentration of 83 mg/l over a period of eight weeks. The resulting findings included occurrence of myelin figures in membrane structures of epithelial cells, occasional disappearance of nuclear membrane, numerous intranuclear cytoplasmic pseudoinclusions, and increased number of perichromatin and interchromatin granules and of nuclear corpuscles. Narrowing of the layer of peripheral condensed chromatin and broadening of the layer of dispersed chromatin were frequently observed. In the cytoplasm of epithelial cells, production of autophagous vacuoles was detected in differentiated types of cells, the presence of great amount of polysomes and profiles of rough endoplasmic reticulum diversely deformed was seen in the undifferentiated neck cells. These cells presented a marked proliferative activity. Some of the changes described in the nuclei of epithelial cells were also observed in different mesenchymal elements in the lamina propria mucosae. The demonstrated changes are indicative of both regressive and progressive effects of the cancerogen used in our experiment.

Acid hydrolases in the extracellular medium of Ehrlich ascites tumor cells.

The hydrolytic activities released to the extracellular medium by tumor cells could be related to the fundamental mechanisms of invasion and metastasis. This paper deals with a study of the extracellular release of several enzymatic activities by Ehrlich ascites tumor (EAT) cells maintained in vitro. The activities of acid hydrolases increase during the first 30 minutes of in vitro incubation to levels significantly higher than those of cytoplasmic enzymes. The presented results are consistent with the hypothesis of the existence of a mechanism in these cells for the specific release of lysosomal contents.

Cobalt-activated acylase from human uterine myoma.

Cobalt-activated acylase was isolated from human uterine muscle and myoma. The enzyme was purified by ammonium sulphate precipitation, and subsequent chromatography on DEAE-cellulose, Sephadex G-150 and DEAE-Sephadex. The comparison of muscle acylase and acylase obtained from myoma has shown differences in the enzyme stability, the dependence of activity on pH and in the susceptibility to the effect of activators and inhibitors. Only one molecular form of cobalt-activated acylase has been found in both tissues.

Chromosomal analysis on Yoshida tumor cells sensitive and resistant to dibromodulcitol.

Yoshida ascites tumor in Wistar rats was rendered resistant to dibromodulcitol. The modal number of chromosomes in the parent line was reduced from 39 to 36 in the resistant one. The parent tumor was a mixed cell population, containing large subtelocentric (LST) chromosome in 65 per cent of the cells, while in the remaining 35 per cent the LST chromosome was absent. In the resistant tumor line, however, the LST marker chromosome was present only in a few cells. The resistant line preserved this new karyotype throughout the 5 months of passaging even without dibromodulcitol, however, parallel to the loss of resistance this tumor line regained the original number of chromosomes, i. e. 39. Selection of a cell population with reduced number of chromosomes seems to be associated to the development of drug resistance.

Combined therapy of leukemia La with Damvar and cytostatics.

The new cytostatic drug Damvar, delta-(2-amino-6-hydroxy-3,4-dihydro-4-oxo-5-pyrimidinyl)valeric acid, is not effective in La leukemia system. Damvar given in combination with 5-fluorouracil enhanced the anti-La effect of 5-Fu. There was also enhancement of the antileukemic effect of suboptimal and optimal doses of cyclophosphamide with the addition of Damvar.

Calculations of theoretical indices of carcinogenic activity including the effect of medium.

Relation between the electronic structure of aromatic compounds and their carcinogenic activity has been correlated. Hypotheses of K, L regions have been employed. Possibilities of better theoretical description of carcinogenic activity have been examined by including the effect of medium and by using a more exact quantum--chemical method. Theoretical carcinogenic activity of some compounds so far experimentally not tested has been estimated.

Inhibitory effect of the antioxidant butylated hydroxyanisole on the activation of the carcinogen benzo(a)pyrene.

The effect of the antioxidant butylated hydroxyanisole (BHA) on benzo(a)pyrene (BP) metabolites binding to DNA, percentage of diols and phenols in the total amount of BP metabolites extracted to organic solvent, were studied in : incubation mixture of rat liver microsomes, cultured mouse embryo cells, human skin, and human lymphocytes. The amount of BP metabolites bound to DNA in mouse embryo cells and human skin decreased in the presence of BHA. No effect was found in human lymphocytes. Percent of phenols increased in mouse embryo cell cultures; percent of diols decreased in cultures of human skin and lymphocytes.

Differences in the purification effect of carcinoembryonic antigen (CEA) from the three different hepatic metastases of rectum carcinoma.

Carcinoembryonic actigen (CEA) was purified from three liver metastases of carcinoma recti by a conventional procedure involving perchloric acid extraction of tumor tissue and fractionation of the extract by gel filtrations on Sepharose 4B and Sephadex G-200. The preparations obtained showed a different degree of heterogeneity in SDS-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis, and also a different antigenic activity. One of preparations obtained was a homogeneous CEA by all these criteria, and two others showed heterogeneity and lower antigenic activity. The most heterogeneous CEA preparation was further purified by the affinity chromatography on Concanavalin A-Sepharose 4B and by DEAE-Sephadex A-50 chromatography and the usefulness of these procedures for purification of CEA is compared.

Changes in hexokinase properties during transformation and subsequent proliferation of liver cells.

The activity and subcellular distribution of hexokinase in liver and several hepatomas were studied. The differences were found in the behavior of this enzyme in spontaneous, primary induced and transplantable hepatomas as compared with normal liver.

DNA methylation in various growth phases of cultured L5178Y murine lymphoblasts.

The extent of methylation of DNA during the transition of cells from exponential phase of growth to the saturation density has been studied in cultured L5178Y mouse lymphoma cells by the comparison of the degree of 5-methylcytosine formation and the stability of this preformed base in various periods of their growth. It has been found that while the degree of 5-methylcytosine formation gradually fell down, the level of this base preformed during the exponential growth was stable during the subsequent periods until the saturation density was reached. At that state some small and variable loss of the base was found.

Comparative in vitro studies of human fibroblastic cells derived from fibrosarcoma, fibroma and various organs of fetus.

Studies on growth of human bladder tumors. The relationship between the proliferative cell population, the chromosome pattern and histology.

Nucleolar morphology in estimation of urinary bladder cancer in man: study on surface contact preparations.

The special feature of nucleolar morphology which reflects the intensity of ribosomal RNA precursor molecule formation and thus also the intensity of proteosynthesis was studied in contact surface preparations from tissue samples of 28 patients with epithelial tumors of the bladder and in tissue samples of bladder mucosa from 16 patients with prostata hypertrophy (7 of them suffered also from cystolithiasis). In malignant lesions of the bladder mucosa, especially in tumors of medium and high grade malignancy, very low values of metabolically resting cells with ring shaped nucleoli and micronucleoli were found.

Histologic changes in the human skin melanoma after intratumorous treatment with BCG.

Twenty six patients with malignant skin melanomas were treated with an intratumorous injection of 0.1 ml BCG applicated in certain time intervals. Histologic changes in tumor tissue starting with dystrophy and necrosis of the cells and ending with the formation of a granular tissue replacing the tumor are described. In one case the untreated satellite localized 2 cm from the injected tumor disappeared. The results indicate that the reaction of the cells on the applicated vaccine varied. In some tumors dystrophic changes and necroses were very large, in others they were of a lesser extent, but in all the process ended with the formation of a granular tissue. The mechanism of the action of BCG is discussed. For the time being, it seems that the intratumorous application of BCG is a useful adjuvant therapy.

Comparative porphyrin content in tumors with contiguous non-neoplastic tissues.

The porphyrin content in primary human carcinomas, their metastases and the contiguous non-neoplastic tissues were compared. The level of porphyrins in primary internal organ tumors was greater, and in endocrine gland tumors lesser, than in their contiguous non-neoplastic tissues. Neoplastic metastases to the liver contained less porphyrin than the host tissue. Metastases both to the liver and to the kidney were characterized by higher pyrrole pigment content than that in the primary tumors.

Organ cultures of embryonic target-tissues exposed transplacentally to chemical substances as a test-system for rapid evaluation of carcinogenicity.

Organ cultures of embryonic target-tissues transplacentally treated by chemical substances are proposed as a test-system for a rapid evaluation of carcinogenicity of environmental pollutions. The results of transplacental effect of some different substances (urethane, polycyclic hydrocarbons, N-nitrosocarcinogens, aminoazo compounds, cyclic amines, aflatoxin B1, pesticide DDT and some noncarcinogenic analogs) on organ cultures of embryonic lung, kidney, liver and brain (hyppocamp) are presented. Transplacental effect in organ cultures depended on the used carcinogen, its dosage, duration of cultivation and on the susceptibility of animals and explanted tissue. The tested carcinogens induced in organ cultures of embryonic target-tissues: 1. specific morphological alterations (diffuse and focal hyperplastic pretumoral alterations of epithelium and tumors); 2. alteration of cell proliferation and 3. an increase of viability and survival of explanted embryonic tissues. This criteria and proposed test-system are critically evaluated with respect to its practical use.