Influence of diabetes on endothelial cell response during sepsis.

AIMS/HYPOTHESIS: Several endothelial pathways of cell adhesion, coagulation and vascular endothelial growth factor (VEGF) signalling are activated during sepsis. The objective of this analysis was to investigate the influence of diabetes on biomarkers of endothelial cell activation in sepsis. METHODS: This was a prospective observational cohort study of a convenience sample of adult patients (age ≥ 18 years) for whom infection was clinically suspected and who presented to an urban tertiary care emergency department between February 2005 and November 2008. We investigated the association of diabetes and sepsis with various endothelial activation biomarkers of cell adhesion (E-selectin, vascular cell adhesion molecule 1 [VCAM-1] and intercellular adhesion molecule 1 [ICAM-1]), coagulation (plasminogen activator inhibitor 1 [PAI-1]) and VEGF signalling (soluble fms-like tyrosine kinase-1 [sFLT-1]). RESULTS: A total of 207 patients (34% with sepsis, 32% with severe sepsis and 34% with septic shock) were studied, including 63 (30%) with diabetes. Compared with patients without diabetes, patients with diabetes had significantly increased E-selectin and sFLT-1 levels overall; this was most pronounced during septic shock in the stratified analysis. Multivariate models including age, sex, sepsis severity and other variables as potential covariates confirmed the association of diabetes with elevated circulating plasma levels of E-selectin (standardised ß 0.24, p < 0.001) and sFLT-1 (standardised ß 0.19, p < 0.01), but there was no significant association with VCAM-1, ICAM-1 or PAI-1. CONCLUSIONS/INTERPRETATION: During septic shock, patients with diabetes had higher levels of circulating biomarkers of endothelial cell adhesion (E-selectin) and VEGF signalling (sFLT-1). Future studies should address whether enhanced activation of the endothelium places patients with diabetes at increased risk for the development of sepsis and worsening morbidity and mortality.

Reversion to normal phenotype induced by SV40 in a spontaneously transformed malignant Chinese hamster cell line.

By using a selection procedure that excluded the transforming effect of SV40, reversions to several properties of normal phenotype were for the first time obtained in a transformed Chinese hamster cell line after SV40 infection. The value of induction to recovery of contact inhibition was typical for SV40-induced reverse gene mutations. Thirteen of 15 isolated revertant clones were T-antigen positive, thus synthesizing the product of viral oncogene. Therefore, in the majority of clones reversion occurred in spite of the presence of viral transforming protein. Dot hybridization revealed the presence of SV40 DNA in all revertants including those expressing no T antigen. The virus rescued from one T-antigen positive and two negative clones proved to be infectious. Reversion to contact inhibition was followed by reversion as regards serum requirements and growth in soft agar. However, in all cases reversion was partial. Karyologic analysis of revertant clones showed that six clones maintained the hypodiploid karyotype of the parental clone, six revertants were near-tetraploid, and one was near triploid. The possible events underlying the SV40-induced reversions to normal phenotype and the role of virus-induced mutations in viral carcinogenesis are discussed.

[Reversibility of malignant transformation as affected by the oncogenic virus SV40. II. The characteristics of virus-induced revertant clones]. / Obratimost' zlokachestvennoi transformatsii pod vliianiem onkogennogo virusa SV40. Soobshchenie II. Kharakteristika virusindutsirovannykh revertantnykh klonov.

Fifteen revertant clones exhibiting contact inhibition, one of the typical characteristics of normal cells, were studied after treatment of spontaneously transformed Chinese hamster fibroblasts with SV40. The clones proved to be partial revertants, as regards to other properties of the normal phenotype--loss of the ability to grow in a medium with a low serum content and anchorage-dependence. Viral DNA was detected in all revertant clones. The expression of T-antigen--the product of viral oncogene, was observed in 13 of 15 revertants analyzed. The study of SV40 "rescued" from several revertants in permissive monkey cells has shown that the virus is non-defective. In 7 clones, reversion was accompanied with polyploidization. In the cases, reversion could be due to changes in the balance between oncogenes and suppressor genes (anti-oncogenes). The possibility of induction by SV40 of mutations in anti-oncogenes suppressing the expression of both cellular and viral oncogenes is discussed. It is suggested that reversion to the normal phenotype in clones with a near-diploid karyotype could result from such virus-induced suppressor mutations.

The action of the tumour promoter, TPA, on mutagenesis induced by different agents (UV light, chemical and viral mutagens).

The present paper deals with effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the frequency of induced mutations to 6-mercaptopurine (6MP) and ouabain resistance in Chinese hamster and mouse cells. UV light, bovine adenovirus 3(BAV-3) and 5-bromodeoxyuridine (BrdU) were used as mutagens. TPA was shown to raise the frequency of gene mutations induced by UV light and BAV-3 but it did not enhance the mutagenic effect of BrdU. We also examined the ability of BAV-3 and BrdU to induce tumours in mice. BrdU was shown to have no carcinogenic effect. The results suggest that TPA enhances the mutagenic effect only for carcinogenic mutagens.

[Reversal of malignant transformation induced by the oncogenic virus SV40. I. Induction of reversal to normal type for the contact inhibition trait]. / Obratimost' zlokachestvennoi transformatsii pod vliianiem onkogennogo virusa SV40. Soobshchenie I. Induktsiia reversii k norme po priznaku kontaktnogo razmnozheniia.

The possibility of induction by the oncogenic DNA-containing virus SV40 of reversions to normal phenotype as regards contact inhibition ("flat" revertants), was studied in spontaneously transformed chinese hamster fibroblasts. Negative selection was used for detection of revertants. The method adopted allowed to study the mutagenic activity of the virus, while excluding its transforming effect. In all experiments the frequency of revertants after infection exceeded that in control series. The value of induction varied from 1.2 to 28.4 X 10(-6). The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) known to increase the frequency of mutations induced by carcinogens in vitro, displayed no enhancing effect on the frequency of revertants induced by SV40. The lack of enhancement of virus-induced reversions after TPA treatment might be explained by the lack of the transforming effect of SV40 in the system studied. Some of the normal "flat" colonies were T-antigen positive, i. e. the viral oncogene was expressed. The role of mutations induced by SV40 in cellular genes controlling malignancy is discussed.

[Integration of SV40 DNA into the cell genome and viral mutagenesis]. / Integratsiia DNK SV40 v genom kletok i virusnyi mutagenez.

Integration of DNA of a temperature-sensitive SV40 mutant (tsA239) into the cell genome was studied. The viral A gene (the oncogene) encodes the tumour T antigen which is ts in the mutant and is devoid of mutagenic and transforming activity under non-permissive conditions (40 degrees C). Clones of Chinese hamster cells infected by tsA239 mutant were analysed. Those infected by wild-type SV40 served as controls. As shown by dot-hybridization, SV40 DNA was detected in cells of 14 out of 18 clones infected by tsA mutant and incubated at 40.5 degrees C, and in all 20 clones infected by tsA mutant and incubated under permissive conditions (33 degrees C), the difference between the two groups being insignificant (p greater than 0.05). By means of blot-hybridization it was established that viral DNA was integrated into the cell genome of all 12 clones analysed, belonging to the three experimental series: infection by tsA mutant, incubation at 40.5 and 33 degrees C, infection by wt SV40, incubation at 40.5 degrees C. The number of integration sites ranged from one to four in different clones. Integration of SV40 DNA in tandems was observed. The data presented allow to conclude that integration per se does not play a crucial role in determining the mutagenic and transforming effect of the virus. Obviously, what matters is the activity of viral oncogene product - the T antigen.

[Mutagenic effect of the SV40 oncogene: induction of resistance to 6-mercaptopurine and serum independence]. / Mutagennoe deistvie onkogena SV40: induktsiia rezistentnosti k 6-merkaptopurinu i nezavisimost' ot syvorotki.

The mutagenic and transforming activity of SV40 DNA fragment, corresponding to its oncogene (the gene for large T antigen) was studied in Chinese hamster cells. After expression time of 3 to 4 days, the oncogene induced mutations of resistance to 6-mercaptopurine (6MP), while the DNA encoding the SV40 late genes, as well as DNA of Chinese hamster cells, were devoid of mutagenic activity. The value of induction ranged from 10(-4) to 10(-5). After the same expression time, the oncogene induced a typical character of oncogenic transformation - independence of serum growth factors (ser+). The value of induction of ser+ variants was somewhat higher than for resistance mutations. The study of 12 clones induced by the oncogene has shown the ser+ character to be hereditary, the expression of viral oncogene being not necessary for its maintenance. The data obtained support the hypothesis in favour of the participation of mutations of cellular genes in viral carcinogenesis.

The oncogene of BAV-3 as a mutagen.

We studied the mutagenic and carcinogenic effects on mammalian cells of two EcoRI DNA fragments of bovine adenovirus type3 (BAV-3) integrated into the pBR325 plasmid. Fragment D located between 3.6 and 19.7 map units, contains the viral oncogene, fragment C, located between 44.3 and 63.7 map units, has no oncogenic activity. The BAV-3 oncogene was shown to increase significantly the frequency of 6-mercaptopurine (6MP)-resistant mutants in Chinese hamster calls. Fragment C, pBR325 without viral sequences and DNA from normal Syrian hamster cells did not have any mutagenic effect. We also looked at the combined action of the viral DNA fragments and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which enhances the transforming effect of carcinogens. TPA was shown to increase the mutant yield on exposure to the viral oncogene but not to induce mutagenic activity in those types of DNA that are unable to transform cells. Probably TPA does not affect the initiation of the mutation process, but acts on later stages just as it affects carcinogenic activity. Thus the results obtained confirm the existence of parallelism between the mutagenic and transforming effects of viral DNA and show that both activities are mapped in the same region of viral DNA - its oncogene.

[Mutagens and the tumor promoter]. / Mutageny i opukholevyi promotor.

We studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and saccharin on the frequency of induced mutations of resistance to 6-mercaptopurine and ouabain in Chinese hamster and mouse cells. UV-rays, bovine adenovirus-3 (BAV-3) and 5-bromdeoxyuridine (BrdU) were used as mutagens. In the case of BAV-3 and BrdU, we investigated, apart from the mutagenic effect, the tumor-inducing activity of these mutagens in mice, BrdU proved to have no carcinogenic effect. The data about the influence of TPA on the mutagenic effect of the three different mutagens indicate that TPA increases the frequency of the gene mutations induced by UV-rays and BAV-3. The results of the study of BrdU and TPA combined action revealed the fact that TPA does not increase the mutagenic effect of BrdU. We demonstrated that saccharin also possesses the promoter activity; it increases the mutagenic effect of BAV-3. The results described above lead to the assumption that TPA influence on the mutagenic effect only takes place when carcinogenic mutagens are used.

[TPA enhancement of the mutagenic and transforming action of bovine adenovirus type 3 on cultured mouse cells]. / Usilenie s pomoshch'iu TFA mutagennogo i transformiruiushchego deistviia bych'ego adenovirusa tipa 3 na kul'tiviruemye kletki myshi.

The cultured 10TI/2C3H mouse cells were infected with bovine adenovirus 3(BAV-3) and treated by TPA. This combined treatment induced ouabain-resistant mutants 19 to 26 times more often than action of only BAV-3. BAV-3- and TPA-treated cells were implanted to syngenic mice. The tumours appeared 10 times more often than in the mice after implantation of only adenovirus treated cells.

[Role of the oncogene in the mutagenic activity of bovine adenovirus type 3]. / Rol' onkogena v mutagennoi aktivnosti bych'ego adenovirusa tipa 3.

The mutagenic and carcinogenic effect of two EcoRI-fragments of bovine adenovirus type 3 (BAV-3) DNA inserted into pBR325 has been studied. The C fragment (located between 3,6 and 19,7 map units) contains the viral oncogene, the C fragment (between 44,3 and 63,7 map units) displays no transforming activity. It has been established that oncogene BAV-3 statistically true increases the yield of mutants resistant to 6-mercaptopurine (6MP) in Chinese hamster cells. The C fragment, pBR325 without viral sequences and DNA fragments of different molecular weights from normal Syrian hamster cells have no mutagenic effect. The control over tumor formation in syngenic mice after injection of C3H10T 1/2 and D. C fragments and pBR325 treatment exposed a parallelism between the mutagenic and transforming effect. The study of the combined effect of viral DNA fragments and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) which increases the transforming activity of different carcinogens, shows that the promoter increases the frequency of mutants after viral oncogene treatment and does not induce mutagenic activity of those types of DNA which are unable to transform the cells.

[Effect of endotoxin on the migration of hematopoietic colony-forming cells and on the repopulation of hematopoietic organs in partially irradiated mice]. / Vliianie éndotoksina na migratsiiu gemopoeticheskikh kolonieobrazuiushchikh kletok i na repopuliatsiiu krovetvornykh organov u chastichno obluchennykh myshei.

In experiments on (CBA X C57BL)F1 hybrids it was shown that the administration of sublethal doses of endotoxin to locally exposed (spleen, 9 Gy) animals enhanced the repopulation of the exposed haemopoietic tissue (spleen and bone marrow). It is concluded that endotoxin has a favourable action on the recovery of haemopoiesis of partially irradiated mice which confirms the idea of the possibility of using stimulators instead of autotransplantation of bone marrow from intact parts.

Mutagenic effects of DNA-containing oncogenic viruses and malignant transformation of mammalian cells.

It was discovered in the 1970s that oncogenic viruses could induce gene mutations in mammalian cells. The phenomenon seems to be widespread: it was observed with all groups of DNA-containing viruses and some retroviruses. The mutagenic effects of the tested viruses at gene level are not locus specific. The viruses induce point mutations, including base substitutions, as well as deletions and insertions. The mutagenic effect of SV40 is controlled by the activity of the early A gene, which encodes the T antigen. Presumably, the process of integration creates the possibility for occurrence of mutations early after infection. Mutagenesis seems to be induced by an integrated virus, though to a much smaller extent. Virus-induced mutagenesis may be connected with an activation of the cell error-prone repair systems. The sum total of the experimental data shows that virus-induced mutagenesis and transformation are interrelated: (A) viruses, like other carcinogenes, display mutagenic activity; (B) viruses that are far removed from each other systematically, whose only similarity lay in being oncogenic and capable of integration, simultaneously showed the ability to induce gene mutations; (C) agents changing the rate of transformation also changed the rate of gene mutations: (D) The function of mutagenicity was mapped in the oncogene of SV40 (gene A); and the DNA of (E) mouse mammary carcinoma virus (MMTV) and avian leukosis virus (ALLV) induced tumors has been found to contain nucleotide sequences that transform 3T3NIH cells but do not carry any viral genetic information. Mutagenesis induced by oncogenic viruses may play a part in the multistage process of malignant transformation, though its contribution may be different in various specific cases and for different groups of viruses. Further studies of the uncommon mutagens, which viruses seem to be, may greatly increase our knowledge of the virus-cell relationship. An understanding of the extent of genetic danger inherent in viruses and live viral vaccines is necessary for practical medicine.

[Malignant transformation of mouse cultured cells using bovine adenovirus type 3 and modification of this process by MNNG]. / Zlokachestvennaia transformatsiia kul'tiviruemykh kletok myshi bych'im adenovirusom tipa 3 i modifikatsiia étogo protsessa MNNG.

It is shown that tumour transformation of cultured mouse cells 10T1/2C3H depends on the time after the infection with virus BAV-3. The maximal number of tumours was observed 3 weeks after infection, then the frequency of tumours was reduced. MNNG modified virus-induced tumour cell transformation differently depending on the time between cell infection and treatment with the chemical agent.

[Genetic nature of one of the traits of malignant cell transformation in vitro]. / Geneticheskaia priroda odnogo iz priznakov zlokachestvennoi transformatsii kletok in vitro.

The genetic events controlling the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied. A hypodiploid clone of Chinese hamster cells with normal serum requirements (49a5ser-) was used as starting material. The results of the fluctuation tests have shown that serum-independence is a random spontaneous event. Its rate of occurrence is 1-2 . 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine) revealed similar characteristics with respect to the distribution of the number of mutants in replicate cultures and the mutation rate. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the oncogenic SV40 virus significantly increased the frequency of ser+ colonies. In the majority of clones isolated in a medium with 1% serum (11 spontaneous and 7 induced by MNNG), the ser+ character proved to be stable after different periods of cultivation without selective pressure. The degree of serum-independence varied in different clones. The results suggest that the ability to grow in a medium with a low serum content originates, in most cases, from a mutation event.

The mutational origin of serum independence in Chinese hamster cells in vitro.

The genetic mechanisms determining the ability of transformed cells to grow in a medium with a low serum content (ser+) were studied in a clone of Chinese hamster cells with normal serum requirements. The fluctuation test has shown that serum independence occurs as a random spontaneous event. Its rate of occurrence is about 10(-5). The concomitant study of a gene mutation (resistance to 6-mercaptopurine--6MP) revealed similar characteristics with respect to the distribution of the number of mutants in replicative cultures. N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) and SV40 significantly increased the frequency of ser+ colonies. Induction was detected after an expression time of 3-4 days, which is typical of gene mutations. In 16 out of 18 ser+ clones of independent origin the ser+ character remained stable. The results suggest that the ser+ character originates in most cases from a mutation event.

The role of the transforming A gene of SV40 in the mutagenic activity of the virus.

The mutagenic activity of the tsA239 mutant of SV40 which synthetizes a defective T antigen at 40 degrees C was investigated in Chinese hamster cells under permissive and nonpermissive temperature. At 33 degrees C the virus increased the yield of 6-mercaptopurine-resistant colonies after 2 days expression time by a factor of 1.6-4 as compared with the control and raised the frequency of aberrant metaphases after the same time by a factor of 1.9-3.4. In the same experiments, with the same initially infected population of Chinese hamster cells, at 40 degrees C tsA SV40 did not induce either gene mutations or chromosome aberrations at the same early stage after infection. Presumably the activity of the A gene of SV40 is necessary not only for the transforming but also for the mutagenic effect of the virus.

Oncogenic adenovirus as mutagen for chinese hamster cells in vitro.

Oncogenic bovine adenovirus (BAV3) was shown to induce chromosome aberrations and gene mutations to 6-mercaptopurine (6MP) resistance in Chinese hamster cells. BAV3 showed the highest mutagenic effect at the chromosome level 12--24 h postinfection. After 48 h the yield of aberrations dropped to the control level, where it remained after 72 and 96 h. BAV3 showed a highly significant induction of mutations to 6MP resistance 48 h postinfection. The effect of the combined treatment of cells with 5-bromodeoxyuridine (BrdU) and BAV3 on mutagenesis at chromosome and gene levels proved to be synergistic. The mutagenic activity at the gene level of BAV3, along with the earlier established mutagenic effect of SV40, indicates that this property is probably inherent in many DNA-containing oncogenic viruses. The possible mechanisms underlying malignant transformation and mutagenesis are discussed.