[The characteristic of immunity in long-lived persons and selection of the immunosenescence markers].

Purpose of the work is to select the indices of immunograms, associated with immunosenescence. The lymphocyte subpopulations were defined by the method of polychrome flowing cytometry in 276 patients of 90+ year with the identical spectrum of chronic unspecific inflammatory diseases. The cluster was revealed consisting of subpopulation of T-lymphocytes (T-lymph) with the high frequency of deviations from standard--helper CD4+, cytotoxic CD8+--T-lymph. The activation of immunity was confirmed by the expression of HLA-DR molecule in 100% of persons with an increase of the absolute number CD4+ and/or CD8+. Reduction in the CD8+ and/or CD4+ was associated with the immune deficient state and the frequency of the increased actTcontent within limits of 63-83%. In 36 long-livers a quantity CD4+ and CD8+ corresponded to standard, 33 of them demonstrated actT exceeded standard. These patients were carried to the group of activation also. Basic group had the quantity CD8+ deficit (73%). In this group, frequency of the development of neuro-degenerate distributions and pathologic changes in the osteomuscular tissue were reliably higher in the persons at the age of 92 years and older than in persons younger 92 years. We consider that CD8+ deficit at the age older 90 year is biomarker of immunosenescence and the risk of an increase in the frequency of development of chronic unspecific inflammatory diseases.

[Influence of various bacterial ligand application methods on cytokine expression].

AIM: Study the production of cytokines in mice during vaccination with polycomponent Immunovac-VP-4 vaccine containing TLR ligands with various administration methods. MATERIALS AND METHODS: Immunovac-VP-4 was administered to mice by subcutaneous, intranasal or per oral methods. The preparation was administered nasally at a single dose of 500 microg in the volume of 30 microl. Per oral single dose was 2000 microg in the volume of 0.5 ml. 200 microg of the preparation was administered subcutaneously. Cytokines in blood sera were determined by EIA 8 hours after the administration of the vaccine. RESULTS: In mice 8 hours afterthe single administration of Immunovac-VP-4 the levels of IL-1beta, IL-6, IL- 12, IL-5 increased significantly. However their concentration differed depending on the method of administration. The most active expression of cytokines was observed during subcutaneous administration. The indexes of cytokine expression were significantly higher (p < 0.05) than during non-parenteral administration methods. CONCLUSION: Mucosal application methods along with parenteral were established to be able to activate effector mechanisms of immune repose with its consequent polarization by Th1/Th2 pathways. These mechanisms lay the groundwork for development of antigen-specific immune responses against antigens/pathogens.

[Assessment of clinical and antiaggregation efficacy of generic clopidogrel 'Egithromb' in roentgenosurgical practice].

The authors studied efficacy of the generic clopidogrel Еgithromb manufactured by EGIS PHARMACEUTICALS (Hungary) based on clinical data and parameters of light aggregometry in a total of 30 patients subjected to planned subcutaneous coronary intervention for stable angina pectoris. It was noted that the generic clopidogrel Egithromb possesses clinical efficacy, manifesting in the lack of early thromboses of the stents and relapses of angina pectoris during the first month of treatment. The findings obtained by light aggregometry strongly suggested a significant decrease in aggregation with a dose of ADP 1.25 mcg/ml in 87.33% of patients, with the target values of the index less than 25% were obtained in 100% of cases. It was determined that the agent possesses high efficacy in aspirin-resistant patients and in the presence of thrombinemia. There were neither side events of therapy nor haemorrhagic complications in the present study during treatment.

[The role of gastric pH and tissue hypoxia in formation of gastroduodenal zone mucose changes in heartsurgical patients].

The estimation of gastric contents pH and gastroduodenal zone mucous membrane condition based on the fibrogastroduodenoscopy data of40 patients who were examined and received proton pump inhibitors in the postoperative period after heart surgical intervention. It was estimated, that acidic peptic aggression and tissue hypoxia are important factors for occurrence of destructive gastroduodenal membrane changes in heart surgery patients. The differences of antisecretory activity of proton pump inhibitors are revealed, so it was useful to use analogous pH-test using in the intensive care unit. The preferences of original omeprazole for intravenous use with it antisecretory effect were marked in comparison with generic omeprazole: the stability of gastric contents pH more than 4,0 was in 100%, more than 6,0 was in 86% of the time for original drug and in 84% and in 27% for generic drug respectively. It is also demonstrated, that cardiotonic drugs using and prosthesis of cardiac valves are additional risk factors of gastroduodenal membrane destruction, independent of acidic aggression.

[A combined method to protect the brain during operations on the brachiocephalic arteries].

Based on the outcomes of surgical management of thirty-six patients diagnosed with atherosclerosis of carotid arteries and a further thirty-five 35 patients presenting with atherosclerotic lesions of the subclavian and vertebral arteries the authors substantiated a differentiated therapeutic policy aimed at protecting the brain from ischaemia, having demonstrated efficiency of cerebral protection with nimodipine and rheopolyglukin in patients subjected to operations on the subclavian and vertebral arteries and feasibility of combining this method with the application of a temporary puncture carotid-artery bypass graft during carotid endarterectomy. Functional possibilities of puncture-mediated bypass grafting of the carotid artery was confirmed by studying the blood flow in the common, internal carotid and median cerebral arteries on the operated side and ipsilateral side in patients with and without a bypass graft applied.

[Assessing clinical efficacy of the generic clopidogrel "Egutromb" in roentgenosurgical practice].

The study comprised a total of ten 47-to-62-year-old (mean age 52.8±2.05 years) patients presenting with coronary artery disease. All patients underwent coronography whose findings were used to determine the indications for stenting of coronary arteries in order to correct coronary insufficiency with due regard for the international guidelines. All the patients received therapy with acetylsalicylic acid at a dose of 75-125 mg/day. As preoperative preparation in all cases the patients were given the generic clopidogrel Egitromb at a daily dose of 75 mg given 120 hours (5 days) prior to the intervention. The clinical effect of the drug was assessed by the presence or absence of relapses of cardiac angina, or the development of acute coronary syndrome within 1 month following the endovascular intervention performed. The state of the vascular-thrombocytic haemostasis was evaluated by the following parameters: spontaneous blood platelet aggregation, induced by ADP given at a dose of 5 µg/ml and 1.25 µg/ml. The obtained initial positive findings of studying clinical efficacy of the generic clopidogrel "Egitromb" manifesting itself in the absence of early stent's thromboses and relapses of cardiac angina within the first month of treatment, as well as in a significant decrease of induced blood platelet aggregation with ADP at a dose of 5 µg/ml and 1.25 µg/ml suggest the necessity of continuing further study thereof in roentgenosurgical practice.

[Diagnosis and treatment of cystic forms of duodenal dystrophia].

Cystic dystrophy of duodenal wall and duodenal dystrophy (DD)--is a rare disease which is based on chronic inflammation of the pancreas tissue (PT), malrelated in the wall of the duodenum (DU). The principal method of surgical treatment of this disease is Pancreaticoduodenectomy (PRD), although it was presented several reports of successful use sandostatin or endoscopic treatment in some patients. Analysis of demographic, clinical and instrumental data, methods of surgical treatment of DD showed that all patients with persistent or recurrent abdominal pain was noted in all patients, weight loss--51%, vomiting--at 26%, jaundice--in 20% of patients. The most accurate diagnostic methods were CT, endo-ultrasound and MRI. The diagnosis of duodenal dystrophy installed in 35 patients. Operations were performed on 22 patients: Implemented PRD (10), removal of pancreatic head resection with a vertical branch of the PT and duodenoduodenoanastomosis (2), pancreatic head resection with excision of the cyst wall of the first portion of duodenum (2), gastric resection (1), resection of the vertical branch of the duodenum with duodenoduodenoanastomosus (2), duodenectomia (1) and resection of the vertical branch of the duodenum with reconstruction of the intestinal insert (2). Four patients fulfilled draining intervention on pancreatic ductal system--pankreatico and cystoenteroanastomoses. Postoperative and late mortality--0.77% of patients the disappearance of pain and 23%--a decrease in its intensity. In two cases, after the PRD against the background of pronounced chronic pancreatitis observed impaired glucose tolerance. Cystic dystrophy of duodenal wall without the expressed "orthotopic" pancreatitis clearly shows pathogenetic, clinical, diagnostic and therapeutic aspects of this disease, causing the possibility of an effective surgical treatment, only limited intervention by the KDP, without resection of the pancreas.

p53 is covalently linked to 5.8S rRNA.

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.

Cytoplasmic p53 polypeptide is associated with ribosomes.

Our previous finding that the tumor suppressor p53 is covalently linked to 5.8S rRNA suggested functional association of p53 polypeptide with ribosomes. p53 polypeptide is expressed at low basal levels in the cytoplasm of normal growing cells in the G1 phase of the cell cycle. We report here that cytoplasmic wild-type p53 polypeptide from both rat embryo fibroblasts and MCF7 cells and the A135V transforming mutant p53 polypeptide were found associated with ribosomes to various extents. Treatment of cytoplasmic extracts with RNase or puromycin in the presence of high salt, both of which are known to disrupt ribosomal function, dissociated p53 polypeptide from the ribosomes. In immunoprecipitates of p53 polypeptide-associated ribosomes, 5.8S rRNA was detectable only after proteinase K treatment, indicating all of the 5.8S rRNA in p53-associated ribosomes is covalently linked to protein. While 5.8S rRNA linked to protein was found in the immunoprecipitates of either wild-type or A135V mutant p53 polypeptide associated with ribosomes, little 5.8S rRNA was found in the immunoprecipitates of the slowly sedimenting p53 polypeptide, which was not associated with ribosomes. In contrast, 5.8S rRNA was liberated from bulk ribosomes by 1% sodium dodecyl sulfate, without digestion with proteinase K, indicating that these ribosomes contain 5.8S rRNA, which is not linked to protein. Immunoprecipitation of p53 polypeptide coprecipitated a small fraction of ribosomes. p53 mRNA immunoprecipitated with cytoplasmic p53 polypeptide, while GAPDH mRNA did not. These results show that cytoplasmic p53 polypeptide is associated with a subset of ribosomes, having covalently modified 5.8S rRNA.

[Stress-dependent lesions of the gastroduodenal mucosa during operations with cardiopulmonary bypass].

A continuous retrospective study was conducted in order to reveal risk factors and optimal ways of prophylaxis of gastroduodenal hemorrhage after operations with cardiopulmonary bypass. All the 559 cases operated on with cardiopulmonary bypass in Department of Cardiosurgery of Omsk Regional Clinical Hospital between January 2003 and December 2005 were analyzed. Risk factors and the effectiveness of different methods of antisecretory prophylaxis of hemorrhage were evaluated. It was found that the frequency of postoperative gastroduodenal hemorrhage was influenced by the degree of circulatory insufficiency, duration of artificial lung ventilation, cardiopulmonary bypass, and aortal occlusion, as well as application of anticoagulants and the frequency of gastric and duodenal erosions or ulcers prior to operation. The most effective means of prophylaxis was omeprazole administered intravenously during three days followed by oral application during three weeks. A short course of H2-histamine blockers discontinued abruptly increased hemorrhage-related lethality in this group of patients due to withdrawal syndrome.

[Peptic ulcers of the gastroduodenal zone in patients with scheduled cardiac operation: continuum of heterogeneous pathogenetic effects].

The article is a review of different pathogenetic mechanisms (infectious, NSAID-associated, stress-dependent and those stipulated by microcirculation disturbances in the gastroduodenal zone) of stomach and duodenum ulcers in patients with scheduled surgical intervention on the heart with the use of cardiopulmonary bypass. Some vexed questions related to treatment tactics of peptic ulcers in these patients are discussed.

[Karyotype characteristics of C3H10T1/2-strain mouse fibroblasts undergoing long-term selection for their capacity to multiply in the presence of ethidium bromide]. / Osobennosti kariotipa fibroblastov myshi linii C3H10T1/2, proshedshikh dlitel'nuiu selektsiiu na sposobnost' razmnozhat'sia v prisutstvii bromistogo étidiia.

Variants Br-0.5 and Br-1 of minimally transformed mouse fibroblasts of C3H10T1/2 line were selected for their ability to proliferate in the medium with 0.5 and 1 mkg/ml of ethidium bromide (EB) toxic for cells of the parent line. Karyological analysis of metaphase chromosomes, stained by Giemsa for G-bands, revealed the number of significant changes in the karyotype of cells resistant to EB. In cells of the resistant sublines the variability of chromosomes was higher than in those of the sensitive population. Two groups of cells are distinguished in the Br-0.5 subline: those with near-diploid and tetraploid chromosome numbers, respectively. The number of polyploid cells in the EB-resistant sublines increases up to 38%, compared to 2% in the parent population. The marker chromosomes in resistant cells originated from translocations, deletions and inversions, with preferential involvement of the material from chromosomes 1.4 and 6. The pericentromeric region of chromosome 4 and the distal region of chromosome I (region 1H1-1H6) were characterized by the increased variability and preferential involvement in rearrangements. In cells of both resistant sublines double mini-chromosomes (1-5 copies per cell) were found. The relation between the revealed chromosomal rearrangements and the mechanism of EB-resistance is discussed.

[Karyotypic characteristics of the murine myeloma line sp2/0-Ag14]. / Kariotipicheskaia kharakteristika myshinoi mielomy linii sp2/0-Ag14.

Using methods of G- and C-banding, a study was made of the karyotype of mouse myeloma cell line sp2/0-Ag14. The number of chromosomes varies from 58 to 65, the modal class being 61-62. 50 per cent of chromosomes are rearranged. Normal chromosomes 6, 12 and X were not detected in either examined cell of this line. Among the marker chromosomes there are an isochromosome (19/19), three dicentric markers and one marker with two interstitial C-bands. There is a specific marker t (12; 15) of mouse plasmacytomas in the karyotype sp2/0-Ag14. A possible association of specific translocations and segregations of the normal chromosomes with the phenotype of line sp2/0-Ag14 is discussed. The results obtained may be useful for cytogenetic analysis of hybridomas.

[Immortalized cell lines from murine embryos are characterized by progressive destabilization of their karyotype structure]. / Immortalizovannye linii kletok, poluchennye iz transgennykh émbrionov myshei, kharakterizuiutsia progressiruiushzchei destabilizatsiei struktury kariotipa.

The karyotype structure was studied for three cell lines obtained from cells of transgenic murine embryos at early stages of their establishment. The first line was obtained from a transgenic embryonic explantant containing oncogen v-sis under promotor MMTV, two other lines originated from cells of transgenic embryos containing oncogen k51. The karyotypic analysis of G-banded metaphase chromosomes revealed deviations from the normal mouse karyotype as early as by the third passage of cultivation of independent embryonic cell lines that contained a foreign oncogene in their genome. The repeated analysis that involved 15-22 passages revealed similar abnormalities: variability and progression in chromosome number with the appearance of hyperpolyploid combinations, and a large number of rearranged chromosomes, both marker and unique ones. It is concluded that introduction of a foreign oncogene into murine cell genome leads to its enhanced and progressive non-specific destabilization. Oncogen v-sis produces a more valuable karyotype destabilization than oncogen k51.

[Chromosomal polymorphism of mammalian cells resistant to drugs in multiple passages. I. Karyotype analysis of Chinese hamster cells resistant to ethidium bromide in the early passages of the first steps of selection]. / Khromosomnyi polimorfizm kletok mlekopitaiushchikh s mnozhestvennoi lekarstvennoi ustoichivost'iu. I. Analiz kariotipa kletok kitaiskogo khomiachka, ustoichivykh k bromistomu étidiiu, na rannikh passazhakh pervykh stupenei selektsii.

G-banded metaphase chromosomes of Chinese hamster V-79 RJK cells resistant to ethidium bromide (2.5 and 10 mcg/ml) have been analysed. The cells of the first selection step (clone IVerb-2, the 9th passage) revealed definite karyotypical instabilities. Amplifications or, in rare cases, deletions were found in chromosomes Z1 and Z6 which appear to have derived from chromosome 1. The amplified region in chromosome Z6 varied considerably in morphology. The chromosome instability, detected in Z1 and Z6, was reproducible in cells throughout the eight independent clones isolated from clone IVebr-2 under non-selective conditions. The data obtained allow to suggest a genetically conditioned mechanism of the above chromosome instability. In the population of resistant cells on the second step of selection (clone I Vebr-5, the 9th passage) the frequency of the cells with amplification increased up to 100%. The length of amplifications increased in the majority of cells. In the cells of the third step of selection (clone IVerb-10, the 12th passage) with near-tetraploid chromosome composition, besides amplifications some specific rearrangements of chromosomes 2 and 7 (markers Z16, Z17) were revealed. The above rearrangements are indicative of the karyotypical destabilization in the drug resistant cells, and may be evaluated as secondary phenomena casually connected with amplifications found at the earlier steps of selection.

[Resistance to ethidium bromide in L929 cells is accompanied by regular changes in karyotypic structure]. / Ustoichivost' k bromistomu étidiiu kletok L929 soprovozhdaetsia zakonomernymi izmeneniiami struktury kariotipa.

The method of differential staining of chromosomes (G- and C-banding) has been used for comparative karyological analysis of mouse fibroblasts of L929 cells selected for resistance to ethidium bromide (EB) at concentrations 1, 10, 25, 50 mkg/ml. All variants have been shown to maintain resistance to EB for a long period of cultivation in nonselective conditions. Only 13 of 36 marker chromosomes of the initial EB-sensitive cells persist, 16 markers being specific for resistant variants. The karyotype changes found occur as early as at the first step of selection and are maintained in variants resistant to the higher drug concentrations. Detection of similar karyotypic changes in clones of independent origin, resistant to EB at concentrations 3 and 25 mkg/ml, allows to conclude that they are quite uniform for L929 cells selected for resistance to EB.

[Karyotypic characteristics of the Chinese hamster cell line CHO-K1 resistant to colchicine]. / Kariotipicheskie osobennosti kletok kitaiskogo khomiachka linii CHO-K1, ustoichivykh k kolkhitsinu.

Karyological analysis was made of G-banded chromosomes in the cells of three independent CHO-K1 clones stably resistant to colchicine (Clch) selected for resistance to Clch at the concentration 0.1 mkg/ml (the first step of selection), and of one clone with higher but unstable level of resistance (2 mkg/ml--the second level of resistance). The results obtained revealed a morphological instability of the P-shoulder of chromosome Z6: more often an additional genetical material (AGM) at the distal end (Z6+), or rarely deletions (Z6-). In cells of the stable resistant clones of the first step of selection the length of AGM and their morphological structures were shown to be constant, but differed among the clones. In cells of the higher resistant unstable clone the length of AGM and their morphological structure were different in different cells within the clone. The AGMs in the Clch-resistant cells are discussed in terms of possible amplification of gene(s) responsible for the Clch resistance. In the cell population of clone selected for the resistance to 2 mkg/ml of Clch the frequency of rearranged chromosomes was shown to increase. In cells of all the analysed resistant clones the chromosome Z16 was found to lose its p-shoulder.

[Karyotype analysis of clone L929 murine fibroblasts by using differential chromosome staining]. / Analiz kariotipa myshinykh fibroblastov klona L929 s primeneniem differentsial'noi okraski khromosom.

Karyological analysis of mouse fibroblasts L929 has been carried out using the differential staining of chromosomes (44-58% of the total chromosome number), and their derivatives, i.e. markers of the particular clone. Normal, non-rearranged chromosomes are mainly present in 1-3 copies, while the markers are available as a single copy only. The frequency of occurrence of diverse chromosomes differs from cell to cell, the total number of chromosomes in the cells being not constant. The modal class consists of 62-64 chromosomes. Two new chromosome markers were found after a repeated karyological analysis one year after the cultivation of cells under the standard conditions. A possible role of some chromosome aberrations in the process of transformation of mouse fibroblasts is discussed. The particular attention is given to alteration of chromosome 15.

[The karyotypic variability of Chinese hamster CHLV-79 RJK cells characterized by multiple drug resistance resulting from the amplification of the mdr gene family]. / Izmenchivost' kariotipa kletok kitaiskogo khomiachka CHLV-79 RJK, kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu, obuslovlennaia amplifikatsiei genov semeistva mdr.

Variability in karyotype structure of Chinese hamster lung V-79 RJK cells and of their six cell sublines, selected for increasing concentrations of ethidium bromide (EB), was investigated in addition to the number of mdr gene copies in cells, both EB sensitive and resistant. It is shown that EB resistant cells exhibit cross-resistance to different drugs resulting from mdr genes amplification. Southern DNA blot hybridization has shown that in Vebr-2 cells (the 1st step of selection) the number of mdr gene copies increased by 10 times, whereas in Vebr-30 cells (the 6th step of selection) the number of mdr gene copies remained the same as in Vebr-2 cells. The level of mdr genes expression in Vebr-30 cells being higher than in Vebr-2 cells. In Vebr-2 cells, homogeneously and differentially stained regions (HSRs) were detected in loci 1p31 and 1q26 of chromosome 1 material (markers Z1 and Z6, respectively). On the following selection steps (prolonged cultivation or increased drug concentration) additional HSRs appeared in chromosome 2 (locus 2qter), in derivatives of chromosome 5 (marker Z7, locus Z7pter) and chromosome X (marker Z2, locus Z2qter). In the course of prolonged cultivation, chromosome 2 and derivatives of chromosomes 1, 2, 5 and X, in which HSRs were found, participated in the formation of new markers resulting from deletions, inversions, insertions and translocations of the chromosomal material. It is supposed that mdr genes amplification in V-79 RJK cells resistant to EB may be regarded as a factor inducing subsequent genome destabilization and eventual progressive changes in the karyotype structure.

[The isolation and genetic characteristics of the new cell line of mouse myeloma spEBR-5 selected for ethidium bromide resistance and characterized by multiple drug resistance]. / Poluchenie i geneticheskaia kharakteristika novoi linii kletok mielomy myshi spEBR-5, otselektirovannykh na ustoichivosti k bromistomu étidiiu i kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu.

Stable mutant cells spEBR-5, resistant to ethidium bromide in concentration of 5 micrograms/ml, have been isolated by multistep selection in mouse myeloma cells sp2/0-Ag14. The spEBR-5 cells, selected with ethidium bromide, appeared to be cross resistant to unrelated drugs in connection with mdr/lb gene amplification and overexpression. Five specific chromosomal markers were detected in the karyotype of spEBR-5 cells as a result of chromosome structural rearrangement. No cytological manifestation of gene amplification such as HCR of chromosomes or DMs was found. A diffuse location of amplified sequences in chromosome(s) is suggested. The new mutant cell lines spEBR-5 can be used as a model for investigation of multidrug resistance mechanisms.