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Numerous areas of the Semipalatinsk Test Site (STS) were subjected to radioactive contamination, including tritium. The tritium content in plants was determined in free water (TFWT) and in the organic component (OBT). It has been established that the OBT content in plants for the "Sary-Uzen" site between combat boreholes ranges from <7-125 Bq/kg and the background OBT content is 21.5 Bq/kg. The maximum OBT values in plants have been recorded in the vicinity of boreholes and range from 7.1 (near the borehole numbered 106) to 200,000 Bq/kg (near the borehole numbered 101). Overall, the tritium content in the vegetation cover at the "Sary-Uzen" site is negligible and does not pose a threat. Isolated cases of tritium content in drinking water exceeding the intervention level have been identified in plants in the "Lazurit" area, as well as near combat boreholes numbered 125 and 101., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The paper provides results of a long-term natural experiment with fruit and berry crops, grown in conditions of radioactive contamination at the territory of the former Semipalatinsk Test Site. The aim of the research is to study peculiarities in accumulation of artificial radionuclides by different species of trees and shrubs. The paper considers only one pathway of the radionuclide's intake, i.e. absorption of the elements by roots from soil. As the result quantitative parameters of 241 Am, 137 Cs, 90 Sr, 239+240 Pu transfer and distribution in different parts of the fruit and berry crops were found. At that radionuclides are not uniformly distributed in plants' organs and in most cases, it obeys the following regularity: leaves > branches > fruits. The difference in accumulation of 137 Cs, 90 Sr, 241 Am and 239+240 Pu for various organs can be range as follows 1.8-77, 1.6-750, 1.9-76 and 3-182 times respectively. The maximum concentrations of 137 Cs in leaves of the researched plants more often were registered at the initial stages of vegetation period. Unlike 137 Cs concentration of 241 Am and 90 Sr in leaves of the researched plants grows usually from the very beginning till the end of the entire vegetation period. The character of transuranium radionuclides accumulation during several years was determined for researched plants. Data obtained can become input parameters for models used to describe the behavior of radioactive substances and assess the risk of contaminated fruit and berry crops for territories having unfavorable radiological situation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The article highlights the results of a radioecological survey of the surface areas of combat boreholes at the Sary-Uzen site located on the territory of the Semipalatinsk Test Site (STS), where underground nuclear tests in these boreholes were conducted earlier. The surface areas of boreholes were examined the levels of radioactive contamination of the soil were determined. A total of 2 main groups were identified. The first group includes boreholes with radioactive contamination of the soil on the surface area. The second group includes boreholes with no radioactive contamination of the surface soil layer. The first group can be divided into boreholes with a deformation of the ground surface and without it. For each variety, depending on the specifics of the nuclear test, the characteristic features of radioactive contamination were determined. Based on the results of the survey, the ranges of specific activity values were established., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The paper provides study of the radioactive contamination of near-surface soil at the Sary-Uzen site in the Semipalatinsk test site (STS). Increased values of natural radionuclides in the studied area were not detected, which indicates the absence of possible geochemical anomalies that could create an elevated radiation background. Radioactive contamination by technogenic radionuclides is caused by two factors. The first factor is fallout of radioisotopes from the atmosphere because of an explosion on September 24, 1951 at the "Experimental field" site. The fallout plume extended from the north-western part of the site to the very south. The range of activity concentration values for 241 Am in soil varies from <0.3 Bq/kg to 390 Bq/kg, with an average value of 5 Bq/kg; for 137 Cs from <1.1 Bq/kg to 330 Bq/kg, with an average value of 55 Bq/kg. A second, but more significant factor is the fallout of radioisotopes due to underground nuclear tests with radioactive release that took place in boreholes on the site itself. The largest area of contamination (about 8 km) is the fallout from testing borehole No. 101 ( 241 Am to 920 Bq/kg and 137 Cs to 12150 Bq/kg)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

This paper reports the speciation of 137 Cs, 241 Am, 90 Sr and 239+240 Pu in the soil samples of the Experimental Field (EF). The EF is a testing ground of the Semipalatinsk nuclear weapons test site used for surface and atmospheric tests. The study revealed low mobility of artificial radionuclides in the EF site soils. The revealed high radionuclide concentrations in soil mainly exist in tightly bound form. On average, the content of the tightly bound form of 137 Cs was revealed to be below 98%, that of 90 Sr - 94%, 241 Am - 89%, and 239+240 Pu - 98%. The radionuclides occurrence forms were analyzed in correlation with the physicochemical parameters of soils. Reliable relationships have been established between the content of carbonates and the content of the exchangeable, acid-soluble and strongly bound 90 Sr forms in soils, as well as the content of the water-soluble salts and the content of the strongly bound 239+240 Pu form in the soil. Similarly, we compared the distributions of the radionuclides speciation and their stable isotopes with their analogous elements in the soil. Unlike 137 Cs and 90 Sr, which are in a tightly bound form in the soils of the Experimental Field site, the main content of soil "competitors" of the 137 Cs radionuclide - K and Cs is observed in an exchange form, less significantly in an acid-soluble form. The alkaline earth metals (analogous elements for 90 Sr) are mainly observed as a composition of the exchangeable and acid-soluble forms. The results allow to conclude that there is no equilibrium distribution of the physicochemical forms of radionuclides introduced into the soil and the natural presence forms of their stable analogs in the soil. Such equilibrium distribution can only be achieved at a complete isotopic exchange in phases and soil components, which under the conditions of the Experimental Field is not possible in the near future. It can be concluded that the behavior of the studied radionuclides in soils is stipulated by the initial form delivered by the fallouts from tests at the EF site., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A study to determine 137 Cs, 90 Sr, 241 Am, 239+240 Pu radionuclides in vegetation cover of the area of craters produced by underground nuclear explosions at the Semipalatinsk test site (STS) is summarised in this paper. Transfer factors (Tf) required for the quantitative description of the radionuclides transition from the soil to aboveground plant parts were found to be highest for sagebrush (Artemisia sublessingiana), Tf values for 90 Sr were gradually decreasing with increasing distance from the dump zone. When arranging the radionuclide transferring factors in descending order, the following sequence was obtained: 90 Sr Tf >  137 Cs Tf >  241 Am Tf >  239+240 Pu Tf, which is consistent with International data. All Tf derived are much higher than those ones derived earlier for epicenters of aboveground nuclear tests and are closest to values for conventionally "background" areas at the Semipalatinsk test site., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This paper provides results of assessment of the tritium distribution in the vegetation cover in the areas of underground nuclear explosions at the Semipalatinsk Test Site (STS). The research was conducted at the former "Degelen" test site along the streams that are one of the main channels of tritium migration from underground nuclear explosions epicenters. The dominant plant species Carex supine and Achnatherum splendens that belong to different ecological groups in relation to humidity were selected as representatives of the vegetation cover. The TFWT (tissue free water tritium) and OBT (organically bound tritium) activity concentrations in the vegetation cover were measurement. TFWT activity concentration in the samples of both plant species had high values with an average of up to 30 kBq kg -1 . The OBT activity concentration was 1-2 orders of magnitude lower than the TFWT in all plant samples. The TFWT and OBT activity concentrations in vegetation samples are closely correlated (r = 0.75, p < 0.05). No statistically significant difference was found between the content of tritium in the samples of Carex supine and those of Achnatherum splendens taken at the same locations. OBT/HTO ratios for vegetation samples of both species were close to equilibrium ratio used in environmental transfer models. In some cases, OBT/HTO ratios were significantly lower than one, which indicates that simple environmental transfer models may not accurately predict the behavior of HTO and OBT in different environmental compartments. The average OBT/HTO ratio for soil samples (0.6 ± 0.1) close to the equilibrium value indicates the equilibrium condition at the research area. The obtained [OBT] plant /[OBT] soil ratios indicate that soil organic matter accumulates tritium from year to year. However, in some locations with high tritium contamination ratios [OBT] plant /[OBT] soil were more than one due to OBT activity in soils is almost the same as OBT activity in plants. It was found that the nature of the spatial distribution of tritium in the vegetation cover in the areas of underground nuclear explosions is complex, and obviously depends on the location of the tunnels in which nuclear tests were conducted, as well as on the peculiarities of the hydrological regime of underground and surface waters, which are the main channels of tritium migration in the research area. Thus, the vegetation cover reflects the spatial distribution of tritium contamination in the sites of underground nuclear explosions and can be used as an indicator of the radiation situation when monitoring radiation-hazardous areas., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A systematic study devoted to 137 Cs, 90 Sr, 241 Am, 239+240 Pu radionuclides in vegetation cover from several spots of the Semipalatinsk test site (STS) is summarised in this paper, highlighting the main findings obtained. The analysed spots are characterized by various types of radioactive contamination. Transfer factors (Tf) required for the quantitative description of the radionuclides transition from the soil to aboveground plant parts were determined, being found that, on average, the minimum Tf for all the radionuclides concerned were determined on the "Experimental Field" ground, followed by the determined ones in the "plumes" of radioactive fallout and in the conditionally "background" territories analysed. The highest transfer factors were characteristic of zones of radioactive streamflows and places of warfare radioactive agent (WRA) tests. On the other hand, ordering the radionuclide transferring factors in descending order, the following sequence was obtained: 90 Sr Tf > Cs Tf >  239+240 Pu Tf >  241 Am Tf, with the 90 Sr Tf, on the average, exceeding the 137 Cs Tf by 8 times and exceeding the 239+240 Pu Tf by up 16 times. 239+240 Pu Tf values were up to 3 times higher than the 241 Am Tf. The exception to the indicated radionuclide Tf descending order corresponded to places of WRA tests where Tf of radionuclides of interest by plants follows the sequence 90 Sr >  239+240 Pu >  137 Cs., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this paper are analyzed the artificial radionuclide distributions ( 137 Cs, 90 Sr, 241 Am, 239+240 Pu) in particle-size fractions of soils from two radioactive fallout plumes at the Semipalatinsk Test Site. These plumes were generated by a low-yield surface nuclear test and a surface non-nuclear experiment with insignificant nuclear energy release, respectively, and their lengths are approximately 3 and 0,65 km. In contrast with the great majority of similar studies performed in areas affected mainly by global fallout where adsorbing radionuclides such as Pu are mainly associated with the finest soil fractions, in this study it was observed that along both analyzed plumes the highest activity concentrations are concentrated in the coarse soil fractions. At the plume generated by the surface nuclear test, the radionuclides are concentrated mainly in the 1000-500 μm soil fraction (enrichment factor values ranging from 1.2 to 3.8), while at the plume corresponding to the surface non-nuclear test is the 500-250 μm soil fraction the enriched one by technogenic radionuclides (enrichment factor values ranging from 1.1 to 5.1). In addition, the activity concentration distributions among the different soil size fractions are similar for all radionuclides in both plumes. All the obtained data are in agreement with the hypothesis indicating that enrichment observed in the coarse fractions is caused by the presence of radioactive particles resulted from the indicated nuclear tests., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This paper provides research data on levels and character of radionuclide contamination distribution in the «sediments- water - plants » system of objects of the Semipalatinsk test site (STS). As the research objects there were chosen water bodies of man-made origin which located at the territory of "Experimental Field", "Balapan", "Telkem" and "Sary-Uzen" testing sites. For research the sampling of bottom sediments, water, lakeside and water plants was taken. Collected samples were used to determine concentration of anthropogenic radionuclides 90 Sr, 239+240 Pu, 241 Am, 137 Cs. The distribution coefficient (K d ) was calculated as the ratio of the content of radionuclides in the sediments to the content in water, and the concentration ratio (F V ) was calculated as the ratio of radionuclide content in plants to the content in sediments or soil., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: To investigate the prognostic value of serum N-terminal pro-brain natriuretic peptide (NT-proBNP) in the development of acute kidney injury (AKI) in patients with acute decompensated chronic heart failure (ADCHF)., Subjects and Methods: Eighty-three patients (55 (66%) men and 28 (34%) women; mean age, 65±11 years) with ADCHF were examined. AKI was diagnosed and classified according to the 2012 Kidney Disease Improving Global Outcomes Clinical Practice guidelines. To rule out contrast-induced AKI, the investigation enrolled only patients in whom radiopague agents had not been injected 7 days before and during hospitalization. Enzyme immunoassay was used to determine serum NT-proBNP concentrations in all the patients upon hospital admission., Results: AKI was diagnosed in 18 (22%) patients, 13 (16%) had Stage I, 4 (5%) had Stage II, and 1 (1%) had Stage III. The serum concentration of NT-proBNP was significantly higher in patients with AKI than that in the other patients [1512.1 (981.0; 2246.2) and 861.8 (499.0; 1383.6) pg/ml (p=0.008). The rise in NT-proBNP concentrations of more than 942 pg/ml was established to be associated with a considerable increase in the risk of AKI (relative risk (RR) was 4.3; 95% confidence interval (CI), 1.27-14.90; p=0.02). RОС analysis indicated that a NT-proBNP level of >942 pg/ml allows prediction of AKI with a sensitivity of 78% (52; 94) and a specificity of 55% (44; 69) (AUC=0.70; p=0.006). Four (5%) patients died in hospital. NT-proBNP levels in all the dead were greater than 942 pg/ml. Two of the 4 deceased patients had AKI., Conclusion: A high level of NT-proBNP in a patient with ADCHF during hospitalization can serve as a biomarker for high risk of AKI and for high mortality rates.

Aim: to investigate the prognostic role of erythropoietin (EPO) in patients with acute coronary syndrome (ACS)., Material and Methods: Eighty-four patients (46 men, 38 women, mean age 63+/-11 years) with ACS were studied. Twenty-one patients had ST-elevation myocardial infarction (STEMI), 12 - non-ST-elevation myocardial infarction, 51 - unstable angina (UA). Ten patients with STEMI received thrombolytic therapy. Coronary angiography during hospitalization was criterion for exclusion from the study. Serum EPO was measured at hospital admission., Results: In-hospital mortality was 5%. Median EPO serum level was higher among patients who died (78.1 [27.8-143.5] vs. 9.0 [6.4-14.1]I.U./ml, p=0.004). Acute kidney injury (AKI) was observed in 7 patients with myocardial infarction (21%) and only in 1 with UA.EPO >10.5 I.U./ml was a significant predictor of AKI development (area under curve 0.73; sensitivity 71%, specificity 67%)., Conclusion: The results of the present study indicate that in patients with ACS admission serum level of EPO may be an important biomarker of development of AKI and in-hospital mortality.

Aim: To investigate the prognostic value of serum endogenous erythropoietin (EPO) in patients with acute coronary syndrome (ACS), including that in the development of acute kidney injury (AKI)., Subjects and Methods: Eighty-four patients (46 men, 38 women; mean age 63 ± 11 years) with ACS were examined. Twenty-one (25%) patents were diagnosed with ECG ST-segment elevation acute myocardial infarction (STSEAMI), 12 (14%) had ECG non-STSEAMI, and 51 (61%) had unstable angina. Thrombolytic therapy was performed in 10 (48%) patients with STSEAMI. The patients whom had not undergone coronarography were included in the investigation to exclude the nephrotoxic effect of X-ray contrast agents., Results: AKI was observed in 7 of the patients with acute myocardial infarction and in only 1 of those with unstable angina. Four (5%) patients died during hospitalization. The EPO level of > 10.5 IU/ml predicted the development of AKI in the ACS patients with a sensitivity of 71% and a specificity of 67%. That of > 13.7 IU/ml was associated with hospital death in the ACS patients with a sensitivity of 100% and a specificity of 75% (AUC = 0.93%)., Conclusion: High serum EPO levels in an ACS patent during his hospital stay may serve as a biomarker for a high risk for AKI and high death rates.

The paper reports on the study of artificial radionuclide accumulation in agricultural crops grown at the territory with high concentration of radionuclides, and first of all - with high concentration of transuranium elements. As a result of this work, peculiarities of accumulation and distribution of artificial radionuclides in the vegetative and generative organs of the studied plants have been revealed. Basic accumulation factors have been found for (137)Cs, (90)Sr, (239+240)Pu, and (241)Am in agricultural products. Accumulation factor dependence on type of planting was found for the investigated types of plants. It has been found that the vegetative organs accumulate radionuclides most of all., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The live attenuated influenza vaccine (LAIV) consists of reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments inherited from the circulating wild-type (WT) parental and the 6 internal protein-encoding gene segments from the cold-adapted attenuated master donor viruses (genome composition 6:2). In this study, we describe the obstacles to developing LAIV vaccine strains depending on the phenotypic peculiarities of the WT viruses used for reassortment. The genomic composition analysis of 849 reassortants revealed that over 80% of the reassortants based on the inhibitor-resistant WT viruses inherited WT NA as compared to 26% of reassortants based on the inhibitor-sensitive WT viruses. In addition, the highest percentage of the vaccine genotype reassortants was achieved when WT parental viruses were resistant to the non-specific serum inhibitors. We demonstrate that NA may play a role in the influenza virus sensitivity to the non-specific serum inhibitors. Replacing NA of the inhibitor-sensitive WT virus with the NA of the inhibitor-resistant master donor virus significantly decreased the sensitivity of the resulting reassortant virus to the non-specific inhibitors.

The live attenuated influenza vaccine (LAIV) currently licensed in Russia consists of the reassortant viruses with hemagglutinin (HA) and neuraminidase (NA) gene segments from the circulating wild-type viruses and the six internal protein-encoding gene segments from cold-adapted master donor viruses (MDV) A/Leningrad/134/17/57 (H2N2) or B/USSR/60/69. Presently, only classical reassortment technique is approved for the generation of Russian LAIV strains. In this work, we describe the obstacles to the development of LAIV 6:2 vaccine strains depending on the phenotypic properties of the wild-type viruses used for reassortment. It was demonstrated that the highest percentage of 6:2 vaccine reassortants could be achieved when wild-type parental viruses were resistant to non-specific gamma-inhibitors. It was shown that it was impossible to generate 6:2 vaccine reassortants possessing six internal genes of the AILeningrad113417/57 (H2N2) master donor virus and avian HA and NA genes from H5N1-PR8 viruses using classical reassortment technique. It was suggested that strong constellation effects between the gene segments of the parental viruses could affect the virus gene reassortment. A strong interaction between the genome segments encoding neuraminidase of avian origin and PB2 gene of PR8 virus was observed. When the PB2 gene was inherited from cold-adapted master donor virus, the neuraminidase was also found to be of MDV origin.

During the period of testing from 1945 to 1962 at the territory of Semipalatinsk test site (STS) within the Degelen Mountains in tunnels, 209 underground nuclear explosions were produced. Many of the tunnels have seasonal water seepage in the form of streams, through which tritium migrates from the underground nuclear explosion (UNE) venues towards the surface. The issue of tritium contamination occupies a special place in the radioactive contamination of the environment. In this paper we assess the level and distribution of tritium in the atmospheric air of ecosystems with water seepage at tunnels № 176 and № 177, located on "Degelen" site. There has been presented general nature of tritium distribution in the atmosphere relative to surface of a watercourse which has been contaminated with tritium. The basic mechanisms were studied for tritium distribution in the air of studied ecosystems, namely, the distribution of tritium in the systems: water-atmosphere, tunnel air-atmosphere, soil water-atmosphere, vegetation-atmosphere. An analytical calculation of tritium concentration in the atmosphere by the concentration of tritium in water has been performed. There has experimentally obtained the dependence for predictive assessment of tritium concentrations in air as a function of tritium concentration in one of the inlet sources such as water, tunnel air, soil water, vegetation, etc.. The paper also describes the general nature of tritium distribution in the air in the area "Degelen"., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Live attenuated cold-adapted influenza vaccine (LIV) has been used in Russia for over 50 years and proved to be safe and effective. Currently, Russian reassortant LAIV is based on influenza AILeningrad/134/17/57 (H2N2) and B/USSR/60/69 Master Donor Viruses (MDVs) which are cold-adapted (ca), temperature-sensitive (ts), and attenuated (att), respectively. The MDVs are used to generate attenuated reassortant vaccine viruses containing the surface antigens of current wild type (wt) influenza A (HINI) and A (H3N2) viruses and wt influenza B virus. The ca/ts/att phenotype of these viruses limits replication in the upper respiratory tract. Reassortment typically yields numerous viruses with different genome constellations, rapid screening is needed to select proper vaccine viruses. In this study, screening of reassortant vaccine strains for live attenuated influenza vaccine generated from currently circulating influenza A and B viruses by RFLP assay is described.

The cold-adapted, temperature sensitive and attenuated influenza master donor viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/ 60/69 were used to generate the vaccine viruses to be included in live attenuated influenza vaccine. These vaccine viruses typically are 6:2 reassortant viruses containing the surface antigens hemagglutinin and neuraminidase of current wild type influenza A and influenza B viruses with the gene segments encoding the internal viral proteins, and conferring the cold-adapted, temperature sensitive and attenuated phenotype, being inherited from the master donor viruses. The 6:2 reassortant viruses were selected from co-infections between master donor virus and wild type viruses that theoretically may yield as many as 256 combinations of gene segments and thus 256 genetically different viruses. As the time to generate and isolate vaccine viruses is limited and because only 6:2 reassortant viruses are allowed as vaccine viruses, screening needs to be both rapid and unambiguous. The screening of the reassortant viruses by RT-PCRs using master donor virus and wild type virus specific primer sets was described to select both influenza A and influenza B 6:2 reassortant viruses to be used in seasonal and pandemic live attenuated vaccine.

Aim: To identify the genes that are responsible for attenuation of donor viruses for live influenza vaccine. MATERIALS AND METHODS. Analysis of phenotypical properties of reassortants of wild type A and B influenza viruses with A/Leningrad/134/17/57 (H2N2) (A17) and B/USSR/60/69 (B60) master donor viruses was performed by comparison of their capability to grow at different temperatures in chicken eggs or/and MDCK cells., Results: Ts phenotype of 178 reassortants of A17 with current non-ts influenza A wild type viruses and 33 reassortants of B60 with current non-ts influenza B wild type viruses were evaluated. Reassortants inherited two polymerase genes PB2 and PA or PB 1 from A17 regularly demonstrated ts phenotype. The polymerase PA and PB2 gene segments of B60 independently controlled manifestation of ts phenotype of B60 based reassortants. The other nonpolymerase genes played no role in manifestation of ts phenotype of reassortants A17 and B60 viruses., Conclusion: The molecular basis for the development ts phenotype of both A and B influenza vaccine reassortant viruses determined by polymerase genes complex.

The cold-adapted (ca) and temperature-sensitive (ts) influenza master donor virus (MDV) B/USSR/60/69 was derived from its wild-type parental virus after successive passages in eggs at 32 degrees C and 25 degrees C. This strain is currently in use for preparing reassortant influenza B vaccine viruses which are used in the Russian trivalent live attenuated influenza vaccine. Vaccine viruses are obtained by classical reassortment of MDV and a currently circulating wild-type virus. The phenotypic properties cold adaptation and temperature sensitivity are inherited from the six genes encoding the internal proteins of the MDV. However, the role of the individual gene segments in temperature sensitivity and thus attenuation is not known. In this study, 35 reassortant viruses of B/USSR/60/69 MDV with current wild-type non-ts influenza B viruses were generated in eggs or MDCK cells and studied in order to identify the genes responsible for their ts phenotype. For each virus the exact genome composition was determined as well as its ts phenotype. The results demonstrated that the polymerase PB2 and PA gene segments of B/USSR/60/69 MDV independently controlled expression of the ts phenotype of B/USSR/60/69 MDV-based reassortant viruses. The other genes coding for internal proteins played no role in this respect. This suggests that mutations in the polymerase genes PB2 and PA play an essential role in attenuation of B/USSR/60/69 MDV-based reassortant influenza B vaccine viruses.

Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.

Screening for candidate reassortants is an important step in the development of live influenza vaccine (LIV). The temperature-sensitive (ts) and cold-adapted (ca) phenotypes of vaccine strains are generally determined, by employing chicken embryos, and used as ts and ca attenuation markers. However, it is difficult to use the egg-determined ts phenotypes of vaccine candidate reassortants as an attenuation marker due to a wide circulation of natural ts epidemic influenza viruses. This study used two new alternative ts and ca attenuation markers in MDCK cells. The MDCK cell line was shown to be able to differentiate cold-adapted influenza viruses from any epidemic strains whereas they were undistinguishable when using eggs. The reduced ability of influenza type A vaccine viruses to grow in the MDCK cell culture at temperatures above 37 degrees C can be successfully used as a "cell-culture" ts marker. The similar marker for influenza B viruses may serve their reduced activity in the MDCK cells at 38 degrees C. The high reproductive activity of cold-adapted viruses in the MDCK cells at 26 degrees C was shown to be a suitable ca attenuation marker. The presented attenuation markers may be included into the standard scheme of primary screening of ts reassortant candidates for commercial live influenza vaccine as additional selection factors and may be used as basic markers in the design of culture vaccine.

The reproducing ability at elevated temperatures (non-ts phenotype) was examined for 38 influenza B virus strains isolated in different years in different countries. Out of the 7 strains isolated in 1940 to 1973, only one showed temperature-sensitivity of reproduction (a ts phenotype). In 1984 to 1988, the proportion of temperature-sensitive strains increased up to 55% (6 of 11). Since the late 1990s, the majority (90%) of the study influenza B viruses demonstrated a pronounced ts phenotype. Influenza B virus strains were also examined for their resistance to serum inhibitors. Prior to the divergence of influenza B viruses into two lines: B/Jamagata and B/Victoria, the epidemic viruses exhibited a high resistance to nonspecific inhibitors of normal equine serum. This property was also preserved in all study B/Victoria strains; however, 83% of the B/Jamagata viruses were inhibitor-sensitive. The present study has demonstrated the heterogenicity of epidemic influenza B viruses in temperature- and inhibitor-sensitivity.

The stability of cold adaptation, temperature-sensitivity, and marker mutations that are typical of attenuated influenza A and B viruses--master donor strains and their based reassortant vaccine strains was studied. After 5 sequential passages in chick embryos (CE) at resolving temperatures of 32 and 37 degrees C, the master donor strains and vaccine viruses retained their adaptability and temperature sensitive phenotype. Passage at the temperatures maximally permissible for viral reproduction (39 and 38 degrees C for influenza A and B viruses, respectively, aborted infection just during the second passage. After a series of passages at all study temperatures), there was neither loss or nor substitution of the marker mutations typical of the cold-adapted and temperature-sensitive phenotype of attenuated viruses. The study supports the high genetic stability of attenuated cold-adapted influenza A and B viruses during CE passage not only at the optimum, but also at elevated incubation temperatures.

Cold-adapted (CA) temperature sensitive and attenuated virus A/Leningrad/134/17/57 (H2N2) (Len/17) has been recently used in Russia as a donor of internal genes in the preparation of reassortant vaccine strains of CA live influenza vaccine (LIV) for all age groups. The Len/17 population was found to be heterogeneous and to be made up of clones, which differ by combinations of mutations in internal genes. Around 50% of the Len/17 population had clones with all 8 coding mutations in internal genes. The others were made up of clones with mutation combinations, which were different from the original Len/17. The PCR restriction method was used to analyze 5 clones of Len/17 and 8 LIV vaccine strains. There were no Ala-86-Thr mutation in the M2 protein in 4 clones and 3 vaccine strains. The PB-1 gene of 4 clones and 3 vaccine strains had a mutation encoding Met-317-IIe more typical of a more attenuated virus A/Leningrad/134/47/57 (H2N2) (Len/47). The NP protein of a clone had a mutation Leu-341-IIe also typical of Len/47. However, neither the absence of mutation in the M2 gene nor an extra mutation in the PB1 gene affected the attenuation extent of reassortant CALIV.

[structure: see text] A strategy to restrict the highly flexible backbone conformation of a peptide nucleic acid (PNA) by incorporation of a cyclopentane ring is proposed. An asymmetric synthesis of cyclopentane-modified PNA is reported, and its binding properties were determined. The cyclopentane ring leads to a significant improvement in the binding properties of the resulting PNA to DNA and RNA.

The influence of ts-phenotype of epidemic viruses and of cold-adapted (CA) reassortant vaccines' strains, appropriately prepared, produced on the human immunogenicity was under investigation. A widespread variability of epidemic viruses' thermal sensitivity sign was established. It was shown that the CA reassortant vaccine strains, obtained through crossbreeding of attenuation donors and of thermally resistant epidemic viruses, are described by a higher immunogenicity. Therefore, the immunogenicity of live influenza vaccines (LIV) can be defined by the ts-phenotype of epidemic parent viruses, which must be sampled for the reassortant vaccine strains not only through searching for samples of antigenically actual viruses but also through search for non-ts-phenotype viruses.

The lipidized derivatives of Bowman-Birk soybean protease inhibitor (BBI) containing one to three oleoyl groups were synthesized and characterized. The (ole)(1)- and (ole)(2)BBI were demonstrated to have 200- and 100-fold higher uptake into Caco-2 cell monolayers compared to native BBI. The acylated BBI had increased affinity to elastase-like proteases. Aprotinin-loaded starch/bovine serum albumin microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride and characterized for their morphology, size and release of the inhibitor. Various formulations of protein proteinase inhibitors were tested for their influence on BHV-1 reproduction in cell cultures. Native aprotinin possessed palpable dose-dependent effect inhibiting the virus reproduction up to 4.0 lg (10,000-fold). The bioadhesive, biodegradable aprotinin-loaded microcapsules were the most effective decreasing virus infectious titer up to 4.0 lg and delaying the cytopathic effect up to 144 h in lesser doses of aprotinin. The lipophilic derivative (ole)(1)BBI was shown to exhibit effective inhibition (>100-fold) of BHV-1 reproduction unlike native BBI.

The objective of this study is to demonstrate the feasibility of microcapsules containing a protein and a proteinase inhibitor in order to allow the oral administration of proteic or peptidic drug. Starch/bovine serum albumin mixed-walled microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride. The microcapsules were loaded with native or amino-protected aprotinin by incorporating protease inhibitors in the aqueous phase during the cross-linking process. Microcapsules can be degraded in the presence of alpha-amylase. The influence of the formulation parameters on the in vitro release of the inhibitor activity and the protein was studied. The protective effect of microcapsules with aprotinin for bovine serum albumin was revealed in vitro. The presence of the native bovine serum albumin was demonstrated after incubation of the microcapsules with aprotinin in a mixture of alpha-amylase (5.4 U/ml) and trypsin (900 spectrophotometric BAEE units/ml) for 3 h at 37 degrees C, whereas the protein was completely degraded in the release medium of the microcapsules without aprotinin.

Preparation of microcapsules through interfacial cross-linking of soluble starch/hydroxyethyl starch and bovine serum albumin (BSA) with terephthaloyl chloride is described. The proteinase inhibitor aprotinin, either native or active site protected, was microencapsulated, being incorporated in the aqueous phase. The influence of aqueous phase pH, BSA, and terephthaloyl chloride concentrations as well as stirring rate on microcapsule morphology and size was studied. The polycondensation pH was shown to be the determining factor for tough microcapsule production with a high encapsulation yield. The size of the microcapsules ranged between 10-30 and 50-100 microm at stirring speed 1500 and 500 rpm, respectively. Fourier transform infrared spectroscopic studies were performed on microcapsules prepared under various conditions. A correlation was established between spectral changes and microcapsule morphology and size. The optimal conditions for microcapsule degradation by alpha-amylase were found. Active site-protected aprotinin was shown to fully retain its activity after microencapsulation.

Autotransfusion of ultraviolet irradiated blood (TUVIB) has been included into the complex of treatment of 96 elderly and senile patients with fractures of the proximal end of the proximal end of the femur. The MD-73M apparatus has been used. The values of central hemodynamics, the threshold of tactile and pain sensitivity, the immune status and the coagulative system of blood have been studied. A high efficacy of TUVIB in this group of the patients has been pointed out, and a conclusion has been made about the necessity of individual choice of the doses of ultraviolet irradiation of autologous blood on the basis of adaptive reaction of calm activation. The side effects and the complications in performing TUVIB, the ways of their prevention and treatment have been considered.