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Nitric oxide (NO) acts as a short-lived paracrine factor and selectively activates transcription of certain genes. The spectrum of inducible genes was studied in primary chondrocytes. A cDNA library was obtained by subtraction hybridization with RNAs isolated from rabbit chondrocytes before and after treatment with nitrosoglutathione, an NO-generating agent. Some of the cloned cDNAs were homologous to known mammalian genes and human EST. NO-dependent transcriptional activation was demonstrated for the stromelysin 1 and cyclooxygenase 2 genes and, for the first time, for mcl1 coding for an apoptosis suppressor.

The response of human and animal cells to the action of fusicoccin (FC), a fungal metabolite with phytohormonal properties, was evaluated. The capacity of FC for inducing the synthesis of early interferon (IFN), supplied into the blood serum of common white mice, and for enhancing the natural cytotoxic activity of human lymphocytes in vitro was established. The metabolism of actively proliferating monocytic leukemia cells J-96 and human ovarian carcinoma cells CaOv, as well as mouse fibroblasts L-929, was found to be inhibited under the in vitro action of FC. The common character of the mechanisms of action of FC and IFN having antiproliferative and immunomodulating activity is discussed.

The response of human and animal cells to the action of fusicoccyne (FC), a fungal metabolite with phytohormonal properties, was evaluated. As shown by in vitro studies, FC had the capacity to induce the production of early interferon (IFN) in the blood serum of non-inbred white mice and to enhance the natural cytotoxic activity of human lymphocytes. In vitro experiments also revealed that the action of FC inhibited the metabolism of actively proliferating monocytic leukemia cells J-96 and human ovarian carcinoma cells CaOv, as well as mouse fibroblasts L-929. The common character of the mechanism of action of FC and IFN, having well-known antiproliferative and immunomodulating activity, is discussed.

Cytokine mRNA expression was studied in human long-term cell cultures of different origin: J-96 and J-41 (monocytic leukemia), SW-13 (paradrenal adenocarcinoma), and MT-4 (T-cell leukemia), in response to IFN-alpha and IFN inducers (kagocel and cycloferon). Cytokine mRNA level in the cell cultures was measured by the RT-PCR method using 11 primer pairs for the following cytokines: IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18 and TNF-alpha. It was shown that IFN-alpha and IFN inducers possess an ability to regulate different cytokine mRNAs. Treatment of the cells with IFN-alpha resulted in expression of mRNAs for IL-2, IL-4, and IL-8. Kagocel induced production of IFN-alpha, IFN-gamma, and IL-2 mRNAs, and cycloferon--IFN-gamma, IL-2, IL-4, and IL-8 mRNAs. It is suggested that antiviral effects of these inducers, in general, can be attributed to imitation of cytokine responses observed in viral infection and, as a result, can lead to starting-up of cellular defense antiviral mechanisms even before action of viruses. Conclusion is made that IFN and IFN inducers may act as regulators of cytokine activity.

Novel approach is suggested to study interferon (IFN) system state in various immunopathological disorders. It is based upon measurement of IFN-alpha and/or IFN-gamma production by leukocytes primed with IFN-alpha or IFN-gamma respectively. Study of IFN-status parameters revealed a tendency to decrease in the induced IFN-alpha production in patients with secondary immune deficiency (SID), autoimmune syndrome (AIS), allergic diseases, lymphoadenopathy (LAP), chronic bronchitis (CB) and bronchial asthma (BAS). Production of the induced IFN-gamma was significantly depressed in all groups except LAP. In priming conditions production of IFN-alpha by leukocytes of the patients with SID, AIS, allergic diseases and LAP increased 1.27-fold (p0.02), 1.31-fold (p0.1), 1.31-fold (p0.001) and 1.27-fold (p0.05), respectively. Under the same conditions production of IFN-gamma by leukocytes of the patients with SID, AIS, allergic diseases, LAP and BAS increased 1.69-fold (p0.001), 2.42-fold (p0.01), 1.63-fold (p0.001), 1.4-fold (p0.1) and 1.69-fold (p0.05), respectively. The data obtained may be useful for understanding mechanisms of the IFN system deficiency development in immunopathological disorders, as well as for further development and improvement of IFN therapy.

Nitric oxide (NO) acts as a short-lived paracrine factor and selectively activates transcription of certain genes. The spectrum of inducible genes was studied in primary chondrocytes. A cDNA library was obtained by subtraction hybridization with RNAs isolated from rabbit chondrocytes before and after treatment with nitrosoglutathione, an NO-generating agent. Some of the cloned cDNAs were homologous to known mammalian genes and human EST. NO-dependent transcriptional activation was demonstrated for the stromelysin 1 and cyclooxygenase 2 genes and, for the first time, for mcl1 coding for an apoptosis suppressor.

Relationship between the priming effects of interferon (IFN)-alpha and -gamma and the standard IFN production by peripheral blood leukocytes was evaluated in 25 volunteers (13 men and 12 women, mean age 35.3 years). Priming of IFN-alpha and IFN-gamma production positively correlated with the standard IFN-alpha and IFN-gamma synthesis, respectively. On the other hand, primed production of IFN-alpha negatively correlated with that of IFN-gamma. Possible role of types I and II IFN interactions in the maintenance of physiological regulatory balance is discussed.

A new approach to evaluation of interferon (IFN) status of patients with acute respiratory viral infections, herpes, and urogenital infections has been developed. It consists in measurement of IFN-alpha and IFN-gamma production by leukocytes primed with IFN-alpha and IFN-gamma, respectively, and subsequent identification of groups of patients whose lymphocytes differ by their in vitro response to IFN priming: sensitive to IFN-alpha and IFN-gamma; sensitive to IFN-alpha but not to IFN-gamma; sensitive to IFN-gamma but not to IFN-alpha; not sensitive to IFN-alpha and IFN-gamma. Such analysis helps understand the mechanisms of IFN system deficiency in infectious diseases and promotes more effective IFN therapy due to selection of patients whose cells retain in vitro sensitivity to a certain IFN type.

The action of mouse serum interferon--alpha/beta (IFN) at the dose of 100 U/ml, of its inhibitor (I) at the dose of 8 U/ml as well as of their combination with the above doses on sensitivities of mouse target cells (TC) of sensitive to IFN line L 929 and resistant to the one line MCB in natural cytotoxic reaction was studied. Cytotoxic activity of human natural killer cells was detected in 14 hrs cytotoxic test using 3H-uridine for labelling of TC. IFN, I, and IFN+I were added to cell cultures for 24 hrs at 37 degrees C with following removing of preparations. It has been shown that I abolished protective effect of IFN on TC L 929 whereas the one possessed the protective action on TC MCB in natural cytotoxic reaction. These data confirmed a suggestion about immunoregulatory properties of I which displayed in abolition or realization of protective effect on TC in natural cytotoxic reaction in dependence on initial sensitivity of TC to antiviral IFN action.

A comparative study of surface membranes of human cell J-96 and J-48 cultures with different sensitivity to alpha/beta interferon (IF). Reduced sensitivity of J-41 cells to IF-alpha/beta was found to be accompanied by a loss of highly specific receptors for IF-alpha, the lack of changes in the cell surface structures upon treatment with IF-alpha/beta, reduced intensity of cell fusion upon successive treatment with IF and polyethylene glycol. The results are discussed in connection with the observed changes in the activity of superoxide dismutase in J-96 and J-41 cell lines after treatment with IF.

The cytotoxic activity of peripheral blood natural killers (NK) against target cells (TC) J-96 and L-929 with high sensitivity to interferon (IFN) action, J-41 and MCB resistant to IFN action and line K-562 labelled by H3-uridine was studied in 14 hrs cytotoxic test. It has been shown that human TC J-96 didn't differ from the J-41 in their sensitivity to NK cytotoxicity and they are strongly resistant to NK than TC K-562. The murine TC L-929 as the human TC didn't differ from the MCB in their sensitivity to NK lysis and had also the same sensitivity to NK as the K-562 cells.

Succinic dehydrogenase activity was studied in the cells of primary cultures of monkey kidney and the HEp-2 strain. As distinct from the primary culture, the activity of the enzyme remains on the same level during the whole period of culturing in the cells of the transplantable strain. The data obtained are discussed in connection with the problem of cell adaptation to the conditions of culturing in vitro.

The effect of human leukocyte interferon (HLI) on three human cell cultures of different origin was studied. HLI inhibited the mitotic activity (MA) of human ovary carcinoma cell culture and to a lower extent that of a diploid cell culture. At the same time HLI preparation exerted a nonspecific stimulating effect on MA of the primary human embryo cell culture. The optimal conditions for the determination of the antimitotic effect of interferon on cells of tumor origin include: a seed dose of 5 X 10(4) cells/ml, addition of interferon to the culture 24 hours after cell seeding; the maximum effect of interferon is observed at 24 hours after addition to the culture.

The results of the study of the antiproliferative effect of swine leukocyte interferon and of an inhibitor of interferon effect in experimental mice with transplanted Krebs-2 ascitic carcinoma cells are presented. The interferon inhibitor exerted antiproliferative effect similar to that of native swine interferon. Combined use of swine interferon and interferon inhibitor did not lead to summation of the antiproliferative effect of these preparations.

Heterochromatin of chromosomes is studied by means of a C-banding technique for the J-96 line of human cells, which is susceptible to enteroviruses and for the J-41 cell line derived from this culture and possessing high specific resistance to Coxsackie B viruses. The data obtained demonstrate stability of variform C-heterochromatin of chromosome pairs 1, 9 16 and certain marker chromosomes in the course of long-term cultivation.

Comparative effectiveness of various methods for production of chronic infection with herpes simplex virus (HSV) in J-96 cell culture: inclusion into the culture fluid of the infected cultures of immune serum to herpes virus, immune serum to HSV-infected J-96 cells and frequent changes of the medium, is described. The advantage of the second method has been found. The chronically infected culture was resistant to superinfection with the homologous virus. In passages, when the chronically infected culture produced no active infectious virus, the portion of cells containing the virus antigen was 0.8%. In this culture interferon was found in a low titer. Morphological and histochemical studies also indicated some changes in chronically infected cultures as compared with the controls, namely, an increase in the number of spindle-shaped elements, thickening of the nuclear membrane, an increase in the DNA concentration. The culture has been designated JH and is in the 220th passage now.

Among 28 strains of influenza A (H3N2) virus isolated from human patients 3 strains were found to be capable of replication in continuous monkey kidney cell cultures. When cells of different origins (PPO, chick embryo chorioallantio membrane, human nasal mucosa) were infected with the virus or when PPO were infected with allantoic or cultural virus strains, the pathological changes were of the same type. No specific inclusions were found in the cells. Adaptation of the virus to PPO cells resulted in inhibition of its hemagglutinating, neuraminidase and interferon-inducing activity and in decrease of thermostability, but the infectious and antgenic properties were retained unchanged.

The role of chromosomes 2, 5, 16, and 21 in production and effect of human interferon was checked in human diploid cells, human heteroploid cells J-96 and clone J-41 thereof. The J-41 cells were found to have a lower number of chromosomes 2 as compared to the other cells under study; J-41 cells produce less interferon than the other cells. Most J-41 cells lack chromosome 21. Unlike the other two cell cultures, these cells do not produce antivirus state after treatment with interferon. The number of chromosomes 16 is larger in J-96 cells than in diploid ones, and they are less sensitive to interferon than diploid cells. The experimental results confirm the importance of chromosome 2 for coding for interferon production, other chromosomes taking part in the regulation of this process. The gene of sensitivity to interferon is localized in chromosome 21, the regulator gene coding the production of repressor of sensitivity to interferon is in chromosome 16.

The application of NORs and R-bands for simultaneous staining of nucleolar organizer regions and banding chromosomes of stable human line is proposed. This technique was applied to studies on the human stable cell line J-96.

The R-method of differential chromosome staining by length was applied to comparative karyological studies on the culture of J 96 human cells susceptible to enteroviruses, and on the J 41 cell line derived from this culture and possessing high specific resistance to Coxsackie B viruses. Karyotype of the J 41 cell line was shown to be deprived of chromosome G21 (P less than 0.0001). The number of other chromosomes varied from cell to cell, but they are constantly present in the majority of cells of both the J 96 and J 41 cell lines. A conclusion is drawn that chromosome G21 incorporates gene(s) which controls the human cells susceptibility to Coxsackie B viruses.

The study was done on a subline of cells reverting to sensitivity to Coxsackie B3 virus after treatment of J-41 cells resistant to this virus with a homogenate prepared from the sensitive J-96 cell culture. Cytogenetic examinations of this cell subline showed its karyological characteristics to approach those of the sensitive J-96 culture. The modal number of chromosomes and the number of chromosomes 2, 9, 11, 12, and 21 were completely restored and marker chromosomes typical of the sensitive culture appeared. In the reverted subline there was almost no marker chromosomes peculiar for the resistant J-41 culture. In addition, a decrease in the number of chromosomes 1 and 19 replicas as compared with J-96 and J-41 culture cells and the presence of marker chromosomes not found in the original cultures indicate that this subline has its own distinctive characteristics.

The antiproliferative (AP) action of human leukocytic interferon (HLI) of varying degrees of purification was studied and compared in cultures of human cells differing in interferon sensitivity. The concentrated and purified preparations of HLI suppressed the incorporation of 3H-thymidine and mitotic activities of the cultures sensitive to HLI of the cells, affecting, but insignificantly, the proliferation of the resistant cell culture. Poorly purified HLI suppressed the proliferation of both cell types. This indicates that native HLI contains impurities that have an AP action and exerts no antiviral activity.

Comparative karyological studies of C-heterochromatin have been made on line J-96 of human cells, which are susceptible to enteroviruses, and on cell line J-41 derived from this culture and possessing highly specific resistance to Coxsackie B viruses. It was shown that the development of specific resistance to Coxsackie B viruses was accompanied by the loss of one of the chromosomes of pairs 1 and 9, and by the dissapearance of two marker chromosomes. There appeared new marker chromosomes with additional C-heterochromatain regions. The data obtained are discussed with respect to a possible interrelationship between these chromosomal alterations and the specific resistance to Coxsakie B viruses.

The cell fusion has been studied in human reticular cell cultures J-96 and J-41 treated with the Sendai virus or with polyethylene glycol 1000 and 6000. The J-96 cells have a high alkaline phosphatase activity, in J-41 cells the enzyme is not detectable. No heterogenous alkaline phosphatase activity was seen in the protoplasm of symplasts 18 hours after virus cell fusion. It has been shown with polyethylene glycol treatment that during the fusion of cells J-96 and J-41 the enzyme activity was spreading over the symplast protoplasm.

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.

A new stable human hypernephroma cell line has been characterized by differential staining R-, C- and Ag-I-techniques. The karyotype of the cultivated hypernephroma cells has been designed using the statistical analysis and karyotype reconstruction methods. The specific chromosomal markers of the cell line are described.

A continuous cell line of human spleen has been obtained. The methods of seeding large numbers of cells and prolonged cultivation of the culture played an important role in isolation of this cell line; both methods have contributed to the creation of conditions for gradual reconstruction of the cellular metabolism in the absence of which the cells wound not be able to get adapted to contiuous cultivation. As the authors failed to obtain a contiuous cell line from a single cell colony the method of "shaking" was applied. The culture was obtained only in case of a 1% PHA solution was added to the nutrient medium.

It is shown that human embryonic cells transformed by Rous sarcoma virus (stable cell line 23) and those transformed by polyoma virus (stable cell line P-2) are morphologically distinguished from the normal human embryonic cells. The mitotic activity of P-2 cells was 51% and the mitotic activity of 23 cells was 48%. While the mitosis activity of human embryo fibroblast was 28%. The duration of the mitosis of P-2 cells was 20 hours and that of 23 cells was 18 hr. The duration of the mitotic cycle of human embryo fibroblast was 18 hr. The G1 periods lasted 6 hours for both the cell lines; the S period of P-2 cells lasted 8 hr and the S period of 23 cells was 6 hr. Both the cell lines had a high content of RNA, DNA, protein bound SH-groups, and a high activity of acid phosphatase, acid RNAase and glucose-6-phosphatase. The content of glycogen, and acidic mucopolysaccharides, the activity of NADPH-tetrazolium reductase, succinic dehydrogenase of both the lines were the same as in normal human cells.