Localization of alpha-fetoprotein in liver tissue of rats during postnatal development: comparison of the immunofluorescent and autoradiographic methods.

Localization of alpha-fetoprotein has been followed in the liver of rats of the Wistar strain from birth up to 37th day of life, and its presence was detected by means of the immunofluorescent and the autoradiographic methods. The former has shown alpha-fetoprotein to be localized in hepatocytes and the number of positive hepatocytes to decline proportionally with that of alpha-fetoprotein concentration in the serum. In newborn rats almost all the hepatocytes were found to be positive. During the course of the subsequent development of the liver tissue the number of positive hepatocytes decreases and at the time of the formation of the lobular structure only certain groups of lobes remain positive. The number of positive cells, gradually diminishes from the periphery towards the vena centralis. The autoradiographic method revealed only small groups of individual cells to be alpha-fetoprotein positive, without any specific localization.

Radioimmunoassay of alpha-fetoprotein in the serum of patients with leukemia and malignant melanoma.

A sensitive double antibody radioimmunoassay has been used to measure Alpha-fetoprotein in the serum of healthy subjects, pregnant women and patients with a variety of malignant diseases including leukemia and melanoma. Elevated serum Alpha-fetoprotein levels were found in 2 of 35 patients with leukemia, 2 of 10 with melanoma. All the pregnant women studied had raised levels.

Enhanced mortality in offsprings of male mice treated with 5-azacytidine prior to mating. Morphological changes in testes.

5-Azacytidine administered to male mice prior to mating resulted in their decreased fertility and in the loss of offsprings at different periods of embryonic and postnatal development. The drug interfered with mature spermatozoa and spermatids; considerable distortion of cellular associations in seminiferous epithelium has been observed. The possible interaction of 5-azacytidine with the function and/or formation of microtubule protein is discussed.

Constitutive heterochromatin in Ehrlich ascites carcinoma cells.

The study of the distribution of constitutive heterochromatin in the chromosomes of Ehrlich ascites carcinoma cells has revealed changes in both the amount and the spatial distribution of this class of DNA. The marker chromosomes were identified and the probable mechanism of their formation was discussed.

Alpha-fetoprotein in the serum of patients with neoplasms of the gastrointestinal tract.

Serum alpha-fetoprotein was estimated in 61 patients with neoplasms of the gastrointestinal tract, 29 healthy controls and 10 pregnant women. Almost half ot the patients had increased values of alpha-fetoprotein and no parellelism between alpha-fetoprotein and liver metastasis was found. Decreasing of the alpha-fetoprotein during the postoperative period could be considered a sign for a total removal of the tumor, and, on the contrary -- increasing of alpha-fetoprotein -- for a recurrence.

Protein histochemistry methods for colloid identification to metastases of thyroid carcinoma.

Evidence for a thyroid origin of tumorous metastases may be facilitated by a histochemical determination of colloid. It is supported by the presence of proteins, demonstrable especially by the reaction for tyrosine, and a simultaneous absence, or only traces of acid mucosubstances. Contrary to existing literary reports, an intraplasmatic formation of mucin has been detected in certain thyroid carcinomas and their metastases. The most difficult to diferentiate histochemically from thyroid colloid is the secrete of certain adenocarcinomas of the prostate.

Uridine kinase: properties, biological significance and chemotherapeutic aspects (a review).

Antibacterial and antitumor activity of some derivatives of ureidosuccinic acid.

The effect of several derivatives of ureidosuccinic acid on the growth of Escherichia coli 387, Staphylococcus aureus strain 209 and its mutant UV-2, Sarcina lutea, Candida tropicalis and Neurospora crossa 9863 as pre-screening systems for antitumor activity was studied. It was found that dihydrazide of D,L-ureidosuccinic acid (DHUA) had a marked antibacterial activity. The inhibitory effect of DHUA on N. crassa could not be removed by aspartic acid, ureidosuccinic acid, dihydroorotic acid, orotic acid, uracil or cytosine. DHUA suppressed the growth of Myeloma P-8 by 38%, that of Sarcoma 180 by 12% and that of Yoshida sarcoma by 19%. No effect was found on the growth of Lymphosarcoma Pliss.

Stepwise transition of aggregate structure of high-molecular-weight avian myeloblastosis virus RNA. Mode of releasing of associated 4S RNA.

Mode of releasing of associated 4S RNA species was studied during a controlled transition of aggregate structure of high-molecular-weight AMV-RNA. It has been found that associated 4S RNA constitutes 2.5% of 60S AMV-RNA complex. Approximately 60% of associated 4S RNA is successively released during treatment of viral RNA with increasing formamide concentration, concomitantly with the transition of 60S RNA aggregate through 50--55S RNA intermediate into the final 30--40S RNA subunits. 40% of 4S RNA remains associated with 30--40S RNA subunits prepared by formamide treatment and can be released from them by heating. A procedure is thus provided both for the isolation of oncornaviral RNA subunit structures deprived of various partions of associated 4S RNA and for the fractionation of 4S RNA species according to their binding affinity to the genome oncornaviral RNA.

Influence of BCG vaccination on the primary and transplantable hamster tumors induced by SV40 and on the specific SV40 virus-induced antitumor immunity.

The main purpose of the study was the estimation of the effect of BCG vaccination and BCG in the combination with the specific immunization with SV40 on the incidence of primary and growth of transplantable SV40-induced tumors in Syrian hamsters. Different BCG vaccine preparations containing 60--100 millions of viable Mycobacteries per 1 mg were used. It has been demonstrated that one and two intradermal injections of BCG (0.25 mg per animal) during the latent period in Syrian hamsters inoculated with SV40 when newborn had no influence on the incidence of primary SV40-induced tumors. Repeated six injections of BCG during the latent period increased the frequency of tumors in one of two experiments. The subsequent was concern with investigation of the effect of BCG vaccination and its different combinations with specific immunization with SV40 on the growth of transplantable SV40-induced tumors in adult Syrian hamsters. Experimental data have shown that different preparations of BCG were varying in the capacity to increase nonspecific antitumor resistance; some preparations were nonimmunogeneic. Injections of a mixture of BCG and SV40 as well as BCG followed with SV40 14 and 20 days later did not lead to summation of effects of immunization, even when immunogenic preparations of BCG were used. Moreover inoculation of SV40 followed with BCG injection 4, 14 or 20 days later completely or partly declined the effect of SV40 specific immunization. Possible mechanisms of the negative effect of BCG on specific antitumor resistance are discussed.

Effect of nucleosides on the synthesis of nucleic acids and proteins in Ehrlich ascites carcinoma cells.

The synthesis of nucleic acids and proteins in whole cells of Ehrlich ascites carcinoma was inhibited by adenosine and by several natural-adenosine-related compounds. The inhibitory effect of adenosine was more pronounced than that of other nucleosides tested (guanosine, inosine, cytidine, uridine and thymidine). The synthetic processes examined were only moderately inhibited by adenine nucleotides (ATP, ADP, AMP, NAD, cyclic 3',5'-AMP and its dibutyryl derivative). Respiration and aerobic glycolysis were not affected significantly by the nucleoside tested.

Immunodiffusion analysis of the antigenic structure of the transplantable MC 29 tumor.

By the use of specific anti-chicken liver sera it could be proved with immunodiffusion techniques that the transplantable MC 29 tumor line contains liver-specific antigens. Production of chicken serum-proteins by the tumor could also be demonstrated. Thus, the transplantable MC 29 tumor line can be regarded as a hepatoma.

Transplantability and biological behavior of a rapidly growing secondary Morris hepatoma.

Transplantable hepatomas have a special importance in cancer research because most are well-differentiated and grow at widely differing rates. A simple and reproducible technique was developed to transplant the rapidly growing Morris hepatoma 9618A2 from a subcutaneous to an intrahepatic site. The tumor has been maintained for over 80 successive transplant generations by inoculating Buffalo rats subcutaneously. The hepatoma, transplanted many times from the subcutaneous site to the liver, was examined histologically on various occasions. By varying inoculum size, growth of the hepatocarcinoma could be controlled. Throughout transplantation, the donor animals were bled without causing the death of the animal. Histological studies revealed that the tumor, when transplanted intrahepatically, remains a highly differentiated neoplasm with no observable morphological changes.

Cell surface antigens detected in cell lines established from lymphomatous Papio hamadryas and Macaca arctoides monkeys.

Cell surface antigens associated with bone marrow cell cultures from leukemic monkeys of species Papio hamadryas and Macacus arctoides were visualized by means of an indirect immunofluorescence method with sera from leukemic baboons. The same immune serum gave two types of immunofluorescence, depending on the origin of the target cells. Fluorescence of the ring-reaction type was seen with Papio hamadryas bone marrow cell cultures growing in suspension and containing the baboon herpesvirus, whereas the patchy type of fluorescence was noted with monolayer bone marrow cell cultures from Macaca arctoides origin, containing type-C oncornavirus but not the herpesvirus particles. Absorption experiments showed that antibodies responsible for the patchy type of immunofluorescence could be absorbed with a disintegrated type-C baboon oncornavirus, and not with baboon lymphoblastoid cell lines containing the herpesvirus nor with human lymphoblastoid cell lines containing the Epstein-Barr virus.

Culture of human cells obtained with DNA from chick Rous sarcoma.

An infectious process was reproduced in the culture of chick embryo cells by means of DNA isolated from Rous chick sarcoma tissue (Carr-Zilber strain). This DNA preparation displays biological activity also in the culture of human embryo diploid cells (HEDC) which is manifested in: 1. discontinuous synthesis of avian oncovirus group-specific antigen; 2. enhancement of proliferative activity and morphological transformation of human cells; 3. continuous presence of virus-specific sequences as revealed by DNA/RNA hybridization. Producing complete oncornavirus by means of DNA isolated from Rous chick sarcoma in HEDC was unsuccessful. DNA preparation from gs negative chick embryo cells shows no infectious activity in HEDC culture.

Humoral antibodies to the capsid antigen of Epstein-Barr virus in Hodgkin's disease.

By the method of indirect immunofluorescence it has been shown in P3HR-I cells that sera from patients with Hodgkin's disease contain high titers of humoral antibodies to the capsid antigen of Epstein-Barr virus (EBV). Higher titers of antibodies of EBV were found in histological variants of Hodgkin's disease with an unfavorable course. The variant of lymphocyte depletion is accomplished by higher titers of the virus and poorer prognosis than the nodular-sclerotic variant having a course with lower titers of antibodies and better prognosis. At the same time, the level of antibodies does not depend on the results of the therapy applied. In the sera of patients with reticulosarcoma or lymphosarcoma no increase in the level of antibodies to EBV has been discovered in comparison with a group of healthy donors; in acute leukemia a certain tendency to decrease in the level of antibodies to this virus can be observed.

A study on the relation between the Epstein-Barr virus and some forms of malignant tumors in children.

The presence of antibodies to the virus capsid antigen of the Epstein-Barr virus was established in the sera of children from different forms of neoplasms with the aid of the indirect method of immunofluorescence according to Henle. 69 sera were studied from children with Wilm's tumor, teratoblastoma, reticulosarcoma, neuroblastoma, sarcoma and also from children with benignant tumors. As control served sera from healthy children of corresponding age. As test cells synthesizing the virus capsid antigen the authors utilized a suspension culture of P3HR-I cells, being one of the clones of Burkitt's lymphoma. The performed investigations have shown that in no one group of children with tumor could there be discovered an increase in the content of antibodies to the Epstein-Barr virus in comparison to controls. It has also been revealed that the spread of the Epstein-Barr virus in different groups of patients and healthy children fluctuated between 83 and 100%.

In vivo blocking of SV40 virus-induced antitumor resistance by Syrian hamster sera from early stages of primary SV40 carcinogenesis.

The blocking of specific SV40-induced resistance of hamsters by the sera collected from 12 individual hamsters infected with SV40 when newborn was studied at different periods of primary virus-induced carcinogenesis. The serum samples were collected at the following periods of primary carcinogenesis: during the latent period (60 days after virus inoculation), at the day of primary tumor appearance, and 19-36 and 45-57 days after primary tumor appearance. For detection of the blocking activity of the collected sera the cells of transplantable SV40 test-tumor were pretreated in vitro with these sera and with control normal hamster sera, and then used for challenge in vivo in SV40-immunized and normal hamsters. With the use of such method, as a rule, no blocking activity of the serum samples collected at any time after the tumor appearance was observed. However, the sera obtained from 7 out of 12 of these hamsters during the latent period significantly decreased the resistance index of animals challenged in transplantation test with the serum pretreated tumor cells. Possible explanations of these findings are discussed.

Varicella-zoster antibodies in patients with Hodgkin's disease.

In an endeavour to find an explanation for th raised incidence of herpes zoster in hemoblastoses, serum samples of 50 patients with histologically confirmed Hodgkin's disease and 80 control sera of normal persons of similar age groups were tested for the presenchods of indirect hemagglutination (IH), complement fixation (CF), and immunodiffusion (ID). The geometric mean titer of IH antibodies and the rate of positive ID results were significantly higher in the patients' group. The geometric mean titers of CF antibodies did not differ significantly in the two groups. A certain correlation between serological results and the histological type of Hodgkin's disease was found, but it was not statistically significant. No other correlation of serological results with clinical data was ascertained.

Histochemistry of oxidoreductases, enzymatic polymorphism and anaplasia of neuroectodermal tumors.

Oxidoreductases were studied histochemically in 162 cases of neuroectodermal tumors. In order of decreasing activity in the cytoplasma these enzymes could be arranged as follows: NADH diaphorase, lactate dehydrogenase, NADPH diaphorase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase. The weak activity of Krebs cycle enzymes and the relatively strong activity of other oxidoreductases, particularly of lactate dehydrogenase, permits to conclude that glycolysis prevails over oxidative processes in neuroectodermal tumor cells. But this should not be interpreted as a decrease of the Krebs cycle enzymes in astrocytoma and oligodendroglioma cells as compared with their parent cells because the latter themselves display a weak activity of these enzymes. A real decrease of Krebs cycle enzyme activity was established only for tumors, the parent cells of which are characterized by a strong (in choroid-papillomas) or moderate (in ependymomas) activity of these enzymes. Many neuroectodermal tumors, in particular those of astrocytic origin, demonstrate a certain correlation between the amount of cytoplasm and oxidoreductase activity. This results in enzymatic polymorphism of the tumor tissue. A certain similarity was established of the oxidoreductase activity in tumor cells and in reactive hypertophic astrocytes. This indicates that both tumor cells and reactive astrocytes may in certain conditions utilize similar mechanisms of increased metabolism. The oxidoreductase activity correlates not with the grade of anaplasia but with different directions of anaplasia reflected in different variants of neuroectodermal tumors. The concept "anaplasia" includes not only certain degrees of dedifferentiation of tumor cells but, as it has been shown histochemically, also an increase of metabolic processes in the tumor cell cytoplasma.

A clinico-pathological study on the correlation of immunosuppression and multiple primary malignant neoplasmas.

At present it is obvious that the incidence of second malignancies in patients with malignant diseases increases after prolonged treatment with immunosuppressive, or antineoplastic agents. The occurrence of such additional malignant diseases was analysed in our five-years autopsy material. During this period 7670 autopsies were performed. Malignant diseases were observed in 1707 cases (22.1%) and among them in 58 cases were proved multiple primary malignant neoplasms (3.3%). The average time between the occurrence of initial and second tumors was 29 months. The frequency of second tumors in patients with leukemias (mainly chronic lymphocytic leukemia) were four times higher than in patients with tumors of epithelial origin. Hepatocarcinomas arose in cirrhotic livers and astrocytomas were often followed by new malignancies. In consequence of successfully applied surgical, radiological and combined immunosuppressive, antineoplastic therapy the survival of cancer patients lengthened, so among different side-effects of the used therapy the oncogenesis cannot be left out of account. The danger of subsequent malignant tumors makes it imperative that immunosuppression should only be applied when strictly indicated.

The levels of NAD and NADH in blood of patients with cancer.

The levels of nicotinamide adenine dinucleotide (NAD) and NADH in blood of patients with cancer in various stages of development as well as their modification after glucose administration were determined in 188 respectively 77 cases. NAD was significantly decreased in patients with cervix and breast carcinoma but unaltered in patients with metastatic cancers (and lung carcinoma) comparatively with healthy subjects. At 48 hours after i.v. administration of glucose, NAD increased significantly in patients with cervix carcinoma but was unaltered in patients with metastatic cancers (an lung carcinoma). NADH in both cases has given little conclusive results.

Nonspecific immunosuppression and expression of avian myeloblastosis virus (BAI strain A).

Chickens were treated with cyclophosphamide in order to induce nonspecific immunosuppression. Treated and untreated animals were injected with avian myeloblastosis virus (AMV) or myeloblasts at the age when a pronounced resistance to the disease is observed. Chickens treated with cyclophosphamide and then challenged with AMV developed acute myeloblastic leukemia in 70 percent. Similarly treated chickens transplanted with fresh AMV producing myeloblasts exhibited 30 percent incidence of myeloblastosis. In contrast, the control animals without treatment showed no myeloblastosis either after myeloblasts application or AMV injection. These results have shown that nonspecific immunosuppression by cyclophosphamide treatment strongly affects the expression of AMV in age-resistant chickens.

Infection of human embryo cells with avian sarcoma virus B77 in vitro.

Human embryo cells were infected with avian sarcoma virus B77--Hu(B77). The virus genome was detected in the Hu(B77) cells during subsequent cell passages in the uncloned cells as well as in single-cell clones. Despite the presence of the virus genome in cells, the growth properties did not differ from uninfected cells. The Hu(B77) cells did not reveal the transformed phenotype in vitro.

An in vitro study of the oncogenic effects of two variants of avian sarcoma virus B77 on rat embryonal fibroblasts.

Two variants of avian sarcoma virus B77 have been tested for their heteroinductive oncogenic in vitro effects on rat embryonal cells. Variant 22-B77V belonging to subgroup B, had been cultured for a long time exclusively on its original host, i.e. on chicks, before being used in the study. This variant of B77V yielded negative results in repeated experiments on rat cells. Variant 55-B77V belonging to subgroup C and which had been made to pass through rat cells, caused a malignant transformation in the latter. The resultant type of this interaction were virus-producing tumorous cells (designated LWF B55). This study present some of the basic characteristics of these cells.

Growth characteristics and tumorigenicity of long-term cultures of the rat RBA myelogenous leukemia.

A continuous cell line, RBA-BC, has been derived from the buffy coat of heparinized blood of a Sprague--Dawley rat with developed RBA myeloblastosis. Separations of myeloblasts from the RBA-BC cell line was accomplished by velocity sedimentation in Verografin density gradients. The cells were monitored during long-term culture by generation time analysis. Cytochemical studies revealed the myelogenous type of RBA leukemia.

Study on the mechanism of interference between Friend leukemia and Sindbis viruses in tissue culture.

Some mechanisms of interference developing in BALB/c mouse embryo fibroblast culture (MEC) infected with Friend leukemia virus (FLV) and 48 hours later superinfected with Sindbis virus (SV) was studied. In FLV-infected cells the amount of SV antigen formed was 2-3 times lower than in SV monoinfection, as indicated by immunofluorescence and cytofluorimetry. Electron microscopic examination showed that in mixed infection the number of newly formed SV particles decreased markedly (by 90%) despite the presence of compact aggregates of viral nucleocapsids in the cytoplasm. When the cells were initially infected with arbovirus and then superinfected with FLV, formation of virus antigen and virions of both viruses was not disturbed. Pre-treatment of cell monolayer with dactinomycin (0.2 mug/ml) blocked interferon production in MEC culture and inhibited interference between FLV and SV. It is assumed that interference between FLV and SV is associated with known mechanisms of interferon action as well as with disturbance of the stage of SV particles assembly and their release from the cell. Due to incomplete cycle of SV reproduction interrupted at the stage of ribonucleoprotein formation, productive type of its interaction with MEC cells is disturbed.

Studies on specific humoral immunity in leukemia.

The paper concerns some results in studies on humoral immunity in human and animal leukemia obtained in author's laboratory (1970-1974). The data presented here disclose immunological mechanisms of mice viral leukemogenesis (cytotoxic and blocking antibodies interrelations). The existence of specific humoral immune response in acute leukemia patients was established (immunoglobulins with the properties of antibodies were revealed in 42.6% of patients). Different forms of specific humoral immunity manifestations in different types of leukemia were distinguished (areactive, nonspecific, cytotoxic, blocking and mixed forms.

Realization of genetic information programming the synthesis of pepsinogen-pepsin in the mucous membrane and tumors of the stomach in man.

Up to 19--20 fractions of separate RNA species and groups have been revealed in malignant tumors of the stomach and mucous membrane of patients with stomach cancer, ulcer and polyposis of the stomach by means of the method of analytical and preparative electrophoresis in 2.5% polyacrylamide gel. A more intensive incorporation of 14C uridine both into the nuclear and separate fractions of cytoplasmic RNA was observed in stomach tumors in comparison with the stomach mucous membrane of man. Pepsinogen-pepsin was synthesized by bound polysoms of the stomach mucous membrane. In stomach malignant tumors of man the polysomes were not capable of synthesizing this enzyme. Fractions of messenger RNA with sedimentation constants 16S-17S, possessing the ability to stimulate pepsinogen-pepsin synthesis in vitro, have been isolated from cytoplasmic RNA of a pig stomach mucous membrane.

Intralesional BCG application in malignant melanoma.

Immunotherapy of malignant melanoma with BCG may be divided into two basic groups: 1. treatment of minimum residual disease. 2. direct intralesional application of BCG. In 19 patients with a histologically confirmed malignant melanoma, direct intralesional application of BCG was used to treat relapsing patients. In 10 of the 19 patients (group A) the relapse was confined to the primary region without signs of distant dissemination. In the remaining 9 patients (group B) signs of the lesion were present prior to BCG application. Our clinical and cytological evaluation bore on local reactions, systemic side reactions and response of non-injected lesions. In patients without signs of distant dissemination, local regression, characterized by a flattening and disappearance of lenticular metastases with scar formation, was achieved in 8/10 patients, while in the noninjected lesions, regression was noted in only 4/10 patients. In 4 patients of group A complete remission lasting 4-6 months was achieved. In the group of patients with signs of distant dissemination, local regression was observed in 6/9, while noninjected lesion regressed in only 1/9. Systemic response to BCG was characterized by febrile reactions with, in the majority of the patients, nausea till vomiting, muscular pain, pain of joints. In the majority of the patients the reaction passed away within 24 hr. A pretreatment with antipyretic and antihistaminic drugs proved of great help. The effect of BCG on the subsequent fate and survival of the patients is not discussed.