Biomedical subjects
Publications and source records attributed to N I Shapiro.
The surviving sepsis campaign developed guidelines in 2003 that were designed to increase physician awareness of sepsis and to develop a series of recommendations for the management of the patient with sepsis. The guidelines had the support of 11 international professional organisations across a variety of specialties, and advocate aggressive, early goal-oriented resuscitation in appropriate patients.
STUDY OBJECTIVE: To determine whether the absolute lymphocyte count (ALC) (white blood count x lymphocyte percentage) can be used to predict a low CD4 count. METHODS: We conducted a retrospective data analysis of consecutive CD4 count analyses performed between January 1, 1995, through December 1, 1995, at an urban university teaching hospital. Results of consecutive CD4 counts and simultaneously measured ALCs were analyzed from samples obtained in inpatient, clinic, and emergency department settings. The ability of ALC to predict a CD4 count less than 200 cells/mm3 was analyzed by calculating sensitivities, specificities, predictive values, and likelihood ratios for a range of ALC values. RESULTS: Among the 807 samples, 322 results (40%) had a CD4 count less than 200 cells/mm3. The ALC and CD4 count were correlated (r=.69, P<.0001). An ALC less than 1,000 cells/mm3 predicted CD4 counts less than 200 cells/mm3 with a sensitivity of .67 (95% confidence interval .62 to .72), specificity of .96 (.94 to .98), positive predictive value of .91 (.87 to .95), and a negative predictive value of .81 (.78 to .84). An ALC less than 2,000 cells/mm3 predicted CD4 counts less than 200 cells/mm3 with a sensitivity of .97 (.95 to .99), specificity of .41 (.37 to .45), positive predictive value of .52 (.48 to .56), and negative predictive value of .95 (.92 to .98). CONCLUSION: A reliable relationship exists between ALC and CD4 count. In a similar population, an ALC less than 1,000 cells/mm3 is predictive of a CD4 count less than 200 cells/mm3, and an ALC greater than or equal to 2,000 cells/mm3 is predictive of a CD4 count greater than or equal to 200 cells/mm3. Physicians may find these criteria useful in identifying patients with increased risk of opportunistic infection.
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.
Four temperature-sensitive HPRT clones were used for hybridological analysis, which led to increase in complementation rate about 5 times. The probability of complementation, in respect of the HPRT locus proved to be rather high: 14 of 45 hybridization-tested mutants had complementation ability (including 3 ts mutants). Analysis of the complementation rate among mutants revealed clear-cut dependence on the selection conditions: clones grown in a medium with 8-azaguanine showed most frequent complementation. The use of mutants with a new phenotype in hybridization analysis revealed four additional complementation groups, three of which are made of temperature-sensitive clones. Biochemical analysis revealed the presence of hybrid forms of the HPRT enzyme in all hybrids tested. This confirms the intragenic character of complementation. At present, the functional map of the HPRT locus is represented by 9 groups, including a group of mutants with no complementation ability.
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.
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 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.
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.
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.
The allergenic action of influenza vaccines was evaluated by the determination of the capacity of sera, taken before and after immunization, for inducing the degranulation of rat mast cells in the presence of the vaccine preparation. In this test sera from three groups of volunteers immunized with experimental and commercial batches of influenza inactivated vaccines, purified and concentrated by gradient centrifugation, was studied. The absence of immediate-type allergization after a single administration of the vaccines under study was shown.
The study deals with intragenic complementation between clones of Chinese hamster cells carrying mutations in the HPRT gene. All clones were of independent origin, selected in media containing one of three purine bases: 8-azaguanine (8 AG), 6-mercaptopurine (6MP), or 6-thioguanine (6TG). Some of the clones were spontaneous, others were induced by various mutagens. To make the study less time-consuming, an experimental set-up was proposed for simultaneous complementation testing of up to 10 clones. As a result, about 400 combinations of clones have been analyzed. Twelve pairs of complementating mutants have been identified in HAT medium. A linear complementation map has been constructed for the HPRT locus, showing five complementation groups. The changes in kinetic and other characteristics observed for mutant HPRT show that all the mutants studied carry structural gene mutations. Analysis of the biochemical characteristics of HPRT has revealed considerable differences between mutant enzymes in clones belonging to different complementation groups (three groups were examined). At the same time, the four mutant clones of complementation group II show similar HPRT characteristics, suggesting a relative similarity of their structural variants of the enzyme. The hybrid nature of HPRT in clones resulting from the fusion of mutant cells confirms the intragenic nature of complementation.
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.
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.
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.
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.
The variants of splitted and subunit influenza monovaccines from virus strains A/Leningrad/385/80R (H3N2) and A/Kiev/59/79R (H1N1), adsorbed on aluminium hydroxide and having the varying content of hemogglutinin and the carrier, have been studied. The immune response of noninbred mice to a single and double injections of these vaccines have been evaluated, the concentrations of the antigen and the carrier inducing a high response in the animals, have been determined. Differences in the immunological potency of hemagglutinins H1 and H3 have been noted.
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.
The materials substantiating the possibility of using the method for the determination of the lethal effect of endotoxin on dactinomycin-treated mice are presented. This determination is made with a view to detecting the admixtures of endotoxins in whole-virion and subvirion inactivated influenza vaccines at different stages of their manufacture, as well as in the final product. The proposed test is highly sensitive, rather simple in its practical realization and can be used for evaluating the degree of the purification of influenza vaccines from endotoxins.