[M-29 human diploid cells line--a substrate for the production of antiviral vaccines].

AIM: Study of morphologic and karyologic characteristics of 5 russian human diploid cell lines (HDC). MATERIALS AND METHODS: 5 HDC lines and HDC strain MRC-5 were studied; RK-13 and Vero continuous cell lines were used; viruses: rubella (RA27/3), measles (L-16), epidemic parotitis (L-3). Cytogenetic analysis of HDC was performed by using DAPI differential staining method. RESULTS: M-29 line has characteristics that are similar to those of MRC-5 diploid cell strain. M-29 cell culture is not contaminated with foreign viruses, mycoplasmata, does not have oncogenic potency. CONCLUSION: M-29 line has high virus-productive properties for accumulation of measles, rubella and epidemic parotitis vaccine viruses and may be recommended as a substrate for the production of antiviral vaccines.

[Treatment of IIIa grade burns with the biologic band].

The biologic band is created on the base of type I collagen and platelet derived growth factor. It was applied for extensive burns of IIIa grade at 1-2nd day after a trauma, which led to epidermis reconstruction at 5-7th day. Authors assume that the effect of the band is determined by the time of its use--1-2nd day, when stimulating exudation qualities combine with healing properties of the bandage. The effectiveness of the collagen band showed to be equal to those of fibroblast transplantation.

[Further studies of continuous human and animal cell lines for the manufacture of viral vaccines and diagnostic kits].

Long-term experiments have provided conditions for the optimal conditions for reproduction of vaccine strains of poliomyelitis, measles, tick-borne and Japan encephalitis on the continuous cell lines. This makes it possible to solve one of the most urgent problems of modern biotechnology, namely to refuse to use primary cell cultures in vaccinology and to apply a more accessible, safe, and reference biological substrate that are stable cell lines.

Characterization of the continuous green monkey spleen cell line 455.

A continuous line of adult green monkey spleen cells has been established and designated 455 (according to the number of animal). Cell suspension for primary explantation has been prepared by perfusion and disaggregation of the organ. The obtained culture, which consisted of fibroblast-like cells with chromosome modal number 60, underwent 25 passages followed by ageing and death (line 455 D). At passage level 13 in 2 culture flasks with 455 D cells, islets of polygonal cells had developed within 35 days; they gave rise to a continuous cell line with chromosome modal number 110 (designated line 455). Line 455 has a high proliferative activity but low demands on the composition of culture media and it can be easily passaged with the bovine serum at concentration of 1-5%. It is highly sensitive to a number of viruses. It is not contaminated with bacteria, fungi, mycoplasmas or viruses, it does not have a tumourigenic activity. Hence, it is a promising cellular model for virological studies, in particular, for preparation of inactivated vaccines along with Vero and 4647 cells. The line 455 can be used for investigation of the problems of haemopoiesis and immunogenesis.

Identification of continuous green monkey spleen cell line 455.

Green monkey spleen continuous cell line 455 has been identified by karyologic analysis, by isoenzyme characteristics and electron microscopy. It has been shown that the line 455 consists of green monkey cells; it is not contaminated by other cells from the green monkey, or by other animal or human cells.

[Delayed disruption of the telomeric links between chromosomes in experimentally induced cells with micronuclei exposed to 5-bromodeoxyuridine in the 1st S period after colcemid administration]. / Zaderzhka raspada telomernykh sviazei khromosom v éksperimental'no indutsirovannykh kletkakh s mikroiadrami pri vozdeistvii 5-bromdezoksiuridinom v pervom S-periode posle vvedeniia koltsemida.

Under a long-term administration of colcemid in the Chinese hamster cell culture some cells with micronuclei are seen to form. In the case of co-treatment with colcemid and 5-bromodeoxyuridine (5-BrdU) at metaphases of the first division of cells with micronuclei polycentric chromosomes were observed. These polycentric chromosomes occur due to delayed disruption of telomeric links, previously existing in the interphase. During colcemid treatment the cells pass through two S-periods: one in mononuclear cells, the other in cells with micronuclei. This phenomenon was tested according to the frequency of metaphases with dicentrics after 5-BrdU-treatment of cells at the first or second S-period or during the two cycles of chromosome replication. The 5-BrdU treatment during the first cycle or two cycles of replication resulted in the same frequency of cells with dicentrics--about 50%. The treatment with colcemid alone during two cycles of replication and administration 5-BrdU at the second S-period results in a considerably lower amount (%) of cells with dicentrics--about 10%. Thus, the delayed disruption of telomeric links between chromosomes may occur under the treatment with 5-BrdU at the first S-period after colcemid administration. It is also concluded that this phenomenon can be reproduced in cell with micronuclei when 5-BrdU is incorporated differentially in the sister chromatids.

[5-Chlorodeoxyuridine, more effectively than 5-bromodeoxyuridine, induces the formation of specific dicentric chromosomes in cells with micronuclei]. / 5-Khlordezoksiuridin bolee éffektivno chem 5-bromdezoksiuridin, indutsiruet obrazovanie spetsificheskikh ditsentricheskikh khromosom v kletkakh s mikroiadrami.

5-chlorodeoxyuridine (5-ChldU) and 5-bromodeoxyuridine (5-BrdU) were tested in respect of the formation of specific dicentric chromosomes in cells with micronuclei. It is shown that the frequency of dicentrics is higher when 5-ChldU is used. On the ground of results of this study and of the previous data it is concluded that the activity of halogenated deoxyuridines is falling with molecular weight increasing.

[Telomeric fusion of the chromosomes in cells with micronuclei during the subsequent incorporation into the DNA of thymidine halogenated analogs]. / Telomernoe sliianie khromosom v kletkakh s mikroiadrami pri posledovatel'nom vkliuchenii v DNK galogenovykh analogov timidina.

As was shown elsewhere among halogenated analogs of thymidine there exist strong (5-chlorodeoxyuridine, 5-ChldU) and weak (5-iododeoxyuridine, 5-IdU) inducers of dicentric chromosomes in cells with micronuclei. Since from colcemid administration to the moment of fixation two cycles of replication are passed, a study was made of the pattern of induction of dicentric chromosomes under subsequent administration of 5-ChldU and 5-IdU, and the other way round. It was shown that the pattern of induction of dicentric chromosomes was strong or weak, and depended on the fact which analog was administered in the first S-period.

[Enhanced intensity of polycentric chromosome formation in cells with micronuclei exposed to 5-bromodeoxyuridine under prolonged hyperthermia at 40 degrees C]. / Povyshenie intensivnosti obrazovaniia politsentricheskikh khromosom v kletkakh s mikroiadrami pri vozdeistvii 5-bromdezoksiuridina v usloviiakh dlitel'noi gipertermii pri 40 gradusov C.

The formation of polycentric chromosomes in metaphases of the first mitotic division of polykaryocytes containing micronuclei, under 5-BrdU treatment in the condition of a prolonged hyperthermia was studied. It was shown that the enhance of the frequency of polycentric chromosomes at 40 degrees C was statistically reliable. These data propose the existence of an enzymatic mechanism of disrupting the chromosomal ends at mitosis at the optimal condition (37 degrees C). In cells with micronuclei, under 5-BrdU treatment at 40 degrees C, this mechanism undergoes, presumably, a partial inhibition leading to the enhanced frequency of chromosomes with fused telomeres.

[An analysis of the chromosomal combinations in dicentrics with induced telomeric fusion]. / Analiz sochetanii khromosom v ditsentrikakh pri indutsirovannom telomernom sliianii.

Chinese hamster heteroploid cell culture was treated by colcemide for 42 h, of which within the first 17 h by 5-bromedeoxyuridine. Dicentric chromosomes, that were formed at the 2nd mitosis after colcemide administration, were analysed using G-banding. Of 211, only 40 were made of heterological dicentrics. Other 171 dicentrics were formed by telomeric fusion of autological chromosomes. No correlation was found between the length, centromeric index and frequency of dicentric formation. A probability of telomeric fusion of chromosomes determined by nucleotide sequences specific for each chromosome is proposed.

[Activity of the nucleolus-organizing regions of the chromosomes of normal and transformed spleen cells in the African green monkey]. / Aktivnost' iadryshkoobrazuiushchikh raionov khromosom normal'nykh i transformirovannykh kletok selezenki afrikanskoi zelenoi martyshki.

The activity of nucleolus organizer regions (NOR's) of chromosomes in the African green monkey cells has been studied before and after the transformation. In cells before their transformation, one or two chromosomes entering into association and possessing NORs are involved in the formation of one nucleolus. In transformed cells no correlation between the numbers of nucleoli in interphase and those of chromosomes possessing NORs was revealed in metaphase plates. It is supposed that in chromosomes of the transformed cells with faintly stained NORs the gene activity is preserved only in part.

[Chromosome nucleolus organizer regions of the African green monkey]. / Iadryshkoobrazuiushchie raiony khromosom afrikanskoi zelenoi martyshki.

The silver staining Ag-I for detection of nucleolar organizer regions (NOR) in the African green monkey chromosomes was used. NOR were situated on the secondary constrictions of one pair of homologous marker chromosomes and consisted of two parts. A block of condensed chromatin was seen in the middle of the despiralized portion corresponding to the secondary constriction. The data suggest that the stalks of satellites of chromosomes, rather than satellites themselves, are regions of nucleolar organizer.

[Spontaneous cytomegalovirus infection of kidney cell cultures of green monkeys]. / Izuchenie spontannoi tsitomegalovirusnoi infektsii kul'tur kletok pochek zelenykh martyshek.

In subcultivation of 104 batches of primary kidney cell cultures from clinically normal green monkeys 66 batches (47%) showed cytopathic changes indicating spontaneous virus contamination of the cultures. Sixty-six strains were isolated and the properties of 23 cytopathic strains were studied. Electron microscopic examinations of 6 virus isolates identified them as members of the herpesvirus group. Serological examinations of 335 clinically normal green monkeys demonstrated antibodies to one of our virus isolates (No. 8532) in 192 (57.3%) animals. Virus strain 8532 was neutralized by antiserum to green monkey cytomegalovirus M-1. The isolates obtained in the present study exhibit the properties previously unknown for green monkey cytomegalovirus such as the capacity for replication in rabbit, sheep, cow, and piglet cell cultures.

[Cultivation of primary animal cells on microcarriers of various types]. / Vyrashchivanie pervichnykh kletok zhivotnykh na mikronositeliakh razlichnogo tipa.

Primary cells of chick embryos (CEC), new-born piglet kidneys (NPK), and green monkey kidney (GMK) obtained by the conventional method of tissue trypsinization were shown to grow satisfactorily on microcarriers of various types. CEC formed a confluent monolayer on Supperbead particles within 38--48 hours when seeded in a dose of 3 x 10(5) cells/ml and on DEAE-Sephadex A-50 within 60--72 hours when seeded in a dose of 4 x 10(5) cells/ml. In both instances, with microcarriers used in a concentration of 1 mg/ml the number of CEC by the end of the cultivation period reached 0.9 million cells/ml. Primary NPK and GMK cells formed monolayers not on all microcarrier particles, nevertheless the number of cells in both cultures increased 2--2 1/2-fold in 7 days. The optimal seed concentration of cells depended both on the quality of the primary cell suspension and on the microcarrier type. The monolayer of CEC cells formed on DEAE-Sephadex A-50 particles persisted for 7--14 days.

[Spontaneous contamination of primary green monkey kidney cell cultures by foamy virus]. / Izuchenie spontannoi kontaminatsii pervichnykh kul'tur kletok pochek zelenykh martyshek peniashchim virusom.

Examinations of 1653 batches of green monkey kidney cell cultures revealed contamination with foamy virus (FV) in 243 (16%) batches. From some of these cultures 65 strains of virus belonging to two serotypes were isolated. Tests on sera from 1122 monkeys revealed antibody to FV in 985 (87.8%) animals. No correlation between the presence of antibody in the serum and virus recovery from monkey kidney cell cultures was observed, however. This might be due either to the lack of the virus in the kidneys or to the failure in its isolation from primary cultures. Passages of cell cultures were shown to facilitate additional recovery of FV. These experimental results may be used for screening and selection of FV-free cultures and for control purposes in the course of vaccine manufacture.

[Creation of an experimental cell culture vaccine against distemper using cell line 4647]. / Sozdanie éksperimental'noi kul'tural'noi vaktsiny protiv chumy plotoiadnykh s ispol'zovaniem kletok 4647.

Experiment in susceptible animals demonstrated the effectiveness and safety of several samples of a distemper vaccine prepared in cell line 4647. Preparations of the vaccine virus variants generated in line 4647 after 10 passages at 34 degrees C, 37 degrees C and 40 degrees C and 20 passages of rapidly multiplying virus (34 degrees C) possessed the protective activity when used for immunization in a dose of 1000 PFU/animal. The results obtained in observations of the animals and examinations of sera for antibodies did not differ from those of control tests of "Vakchum".

[Comparative analysis of the virus-specific proteins of cells infected with untypable herpes simplex virus strains]. / Sravnitel'nyi analiz virusspetsificheskikh belkov zarazhennykh netipiruiushchimisia shtammami virusa prostogo gerpesa kletok.

Herpes simplex viruses of the so-called intermediate types were typed by analysis of virus-specific proteins of the infected cells. The results obtained correlate well with the results of typing of these strains by DNA analysis using restriction endonucleases. Strain differences of virus-specific proteins of herpes simplex virus of both types were established.

[Comparison of methods for the large-scale culture of cells and viruses]. / Sravnenie metodov krupnomasshtabnogo kul'tivirovaniia kletok i virsuov.

The paper presents the results of experiments on propagation of primary, secondary, and continuous diploid and heteroploid human and animal cells in 2 different systems for large-scale propagation: in perfusion tank with Rashig rings and in tanks with microcarriers. Both methods of large-scale cultivation produce higher cell yields than the traditional cultivation methods. A yield of tick-borne encephalitis virus per 1 cell in the perfusion cultivator was 12 times as high as in roller cultures. Poliomyelitis virus titres were practically equal with both methods of large-scale cultivation and with the conventional method (7.73 lg PFU/ml in GMKC on microcarriers). The method of cultivation on microcarriers is more acceptable and advantageous as it gives higher yields of cells necessary for growth of poliomyelitis virus.

[Cultivation of the rabies virus in the continuous kidney cell line 4647 from the green marmoset]. / Kul'tivirovanie virusa beshenstva v linii perevivaemykh kletok pochki zelenoi martyshki 4647.

Continuous cells of green monkey kidney 4647 were found to be highly sensitive to three variants of the Vnukovo-32 vaccine strain of rabies virus preliminarily adapted to the primary line of Syrian hamster kidney cells. The virus litre increased with passages in the 4647 cell culture. The resulting virus variants had sufficient immunogenic potency. The time course of virus replication at a temperature of 32 degrees C was studied. The heteroploid cell line 4647 was found to be promising for manufacture of medical and veterinary cultural rabies vaccines.

[Interferon status and the activity of dsRNA-dependent enzymes in volunteers and influenza patients receiving human recombinant alpha 2-interferon (reaferon)]. / Interferonovyi status i aktivnost' dsRNK-zavisimykh fermentov u dobrovol'tsev i bol'nykh grippom liudei, poluchavshikh chelovecheskii rekombinantnyi al'fa 2-interferon (reaferon).

The national recombinant preparation of alpha 2-interferon (reaferon) after intramuscular inoculation in a daily dose of 3 X 10(6) units/ml to human volunteers was found in the blood serum within 1-2 hours. In 85% of the subjects given the preparation, a 2-8-fold rise in interferon titers in the blood serum and in 64% an increase in interferon leukocyte reaction (ILR) by the 5th day after the beginning of the treatment were observed. In the examined influenza patients given the preparation, interferon circulation in the blood serum, increased ILR and 2-5A synthetase enzyme levels were demonstrated. Tests for interferon of the blood sera of patients given reaferon by inhalation revealed no significant rise in its level, in contrast to cases with reaferon intramuscular injections. It is assumed that the immunomodulating effect of the preparation given by inhalation is mediated through the hypothalamus. Thus, parameters of the interferon system may be corrected by administration of reaferon.