[The application of CytoDiff for the differential diagnosis of reactive and tumor lymphocytosis.] / Применение реактива CytoDiff для дифференциальной диагностики реактивного и Ð¾Ð¿ÑƒÑ Ð¾Ð»ÐµÐ²Ð¾Ð³Ð¾ лимфоцитоза.

CytoDiff reagent was developed to optimize the performance of a WBC differential evaluation. It determines the main population of white blood cells by the expression of linear markers. One of the additional possibilities of using CytoDiff is the differential diagnosis of reactive and tumor lymphocytosis. We studied peripheral blood samples of 76 patients of the All-Russian Central Research Center A.M. Nikiforova EMERCOM of Russia with absolute lymphocytosis of more than 3,0x109/l. The control group included 26 practically healthy people. All performed a clinical blood test on a 5Diff hematology analyzer with smear microscopy, and determination of leukocyte populations by phenotype using CytoDiff. Reference intervals were determined for subpopulations of lymphocytes using CytoDiff. An algorithm has been developed for evaluating the results obtained when determining leukocyte populations by phenotype using CytoDiff for differential diagnosis of reactive and tumor lymphoproliferation. To detect B-cell lymphoproliferative diseases, the use of a cut-off value of 13% or more of the number of leukocytes is optimal. At low values of the relative number of B-lymphocytes, it is important to take into account the results of microscopy of blood smears. If atypical mononuclear cells are absent in smears, then additional clinical and laboratory studies are necessary to establish the cause of lymphocytosis, including phenotyping of peripheral blood lymphocytes to exclude T-cell lymphoproliferative diseases. The expediency of using the CytoDiff reagent for the differential diagnosis of the reactive and tumor nature of lymphocytosis is shown. Already at the stage of primary screening studies, the use of CytoDiff makes it possible to efficiently collect blood samples from patients with possible lymphoproliferative diseases, which significantly reduces the time required for a diagnostic search.

Results of the simultaneous measurements of radon around the Black Sea for seismological applications.

Results of measurements of radon around of the Black Sea are shown. Radon stations in zones of active faults were placed. Simultaneous hourly measurements of soil radon in 2005 were carried out in the Sivrice Fault Zone that is a segment of East Anatolian Fault System, in the town of Tbilisi (Georgia) and in the South Russia. In 2008 simultaneously hourly measurements of soil radon were carried out in the Western Caucasus (Russia) and in the Mytilene Island (Greece). In 2013 radon in underground waters simultaneously in midday was measuring in Crete (Greece), in the Pamukkale geothermal region (Southwest Turkey) and in the Western Caucasus. Measurements of radon concentration in the points located around of the Black Sea have shown identical regularities in changes of the data. Influence of meteorological, tidal and solar factors on changes of water radon concentrations and soil radon concentrations was observed in all researches points. But this influence was insignificant. Seismological application of observed results also was considered. Various mathematical methods of definition of anomaly in the radon data during earthquakes were considered. During researches in the Black Sea region basically earthquakes with M from 2.0 up to 5.0 and in a depth about 10 km were occurred. For these earthquakes method of daily subtraction of the data of the next and previous day was used. This method has allowed solving a problem with a choice of average value. Probability up to 0.69 (number of earthquakes with radon anomalies/total number of earthquakes) of detection of radon anomalies before earthquakes was achieved applying this method. Changes of radon maps before regional earthquakes were also observed. The frequency analysis of variations of the radon data on the basis of the Wavelet analysis was carried out. Occurrence of the short periods (about 2 days) was observed during regional earthquakes.

Effects of the copy number of ribosomal genes (genes for rRNA) on viability of subjects with chromosomal abnormalities.

The number of active ribosomal genes (AcRG) was evaluated in 172 carriers of chromosomal abnormalities (CA) such as Down's syndrome (DS), Robertsonian translocations (RT), Klinefelter's and Turner's syndromes, trisomy Ð¥, disomy Y, and various structural CA. In controls (n=318), AcRG dosage varied from 119 to 190 copies with a mean of 151 copies per diploid genome. In CA carriers, except for DS newborns, AcRG dosage was not beyond these limits. As shown previously, only within these limits cellular homeostasis and organism's viability can be supported, while genomes beyond these limits are eliminated by embryonic loss. About 10% of embryos with DS and 50% of embryos with RT die/are aborted exclusively due to a surplus (DS) or a shortage (RT) of AcRG. AcRG dosage also affects the CA carrier's viability after birth, as demonstrated by comparing newborn and aged (10-40 y.o.) DS patients. Sampling range of AcRG dosage becomes considerably narrower with age: DS newborns ranged from 139 to 194 RG copies (σ2=3.59), while aged DS patients varied from 152 to 190 copies (σ2=1.55) with the same mean. Each CA group showed peculiarities in AcRG dosage distribution. We found that carriers of numerical abnormalities of gonosomes (sex chromosomes) concentrate within the area of medium, most adaptive dosages, whilst carriers of structural CA can only survive with relatively high AcRG number. Our article is the first ever to report an association of CA viability with the genomic number of AcRG.

Measurement of radon in ground waters of the Western Caucasus for seismological application.

Results of radon ((222)Rn) concentration measurement in ground waters in the Western Caucasus are described. In 2010-2011 each day at 12:00 Moscow winter time (9:00 GMT) sampling in volume of 0.5 l of waters was carried out in two wells at depth of 30 m and 180 m. In 2012 three times per day (7:00, 12:00, 17:00) this sampling was already carried out. Radon from water was extracting by degassing method with use of bubbler. Measurements of alpha activity of gas in scintillation (ZnS) chambers were done. The water radon data with seismic, meteorological and the Sun-Lunar data were compared. The mathematical method of definition of "splashes" in radon data before regional earthquakes is considered. The greatest probability in 72% of the forecast of regional earthquakes for the data from a well of 30 m depth was received. Correlation between meteo and radon data is absent. Correlation of lunar phases and solar activity with radon data is discussed. In July-December, 2012 sampling of water from 15 wells and measurements of radon were carried out. The distance between wells was near 50 km. Changes of radon maps in territory of South Russia during earthquakes are shown.

[Primary immunodeficiencies are a topical problem of modern medicine].

AIM: To analyze the incidence of primary immunodeficiencies (PIDs), to reveal the specific features of the course of this condition at the present stage, and to estimate the quality of health care to patients with PIDs. SUBJECTS AND METHODS: An open-label prospective trial was performed in 94 patients with different forms of PIDs (63 with selective immunoglobulin A (IgA) deficiency and 31 with other more severe primary immunodeficiencies) who had been permanent residents in the Perm Territory in the period 1990 to 2012. RESULTS: The registered PID cases were noted to be lower than the estimated ones. Over 22 years of follow-ups, the death rates for this group of patients with these diseases were 11%, and the disability rates were 27%. In severe PIDs (exclusive of selective IgA deficiency), these rates were as high as 35.5 and 96%, respectively. The rate of untimely diagnosis of severe PIDs was high (43%). Molecular genetic studies were conducted in only one tenth of the patents with this disease. PID treatment generally complied with the accepted medical standards. However, all patients with X-linked agammaglobulinemia were observed to have periodic irregularities of replacement therapy with intravenous immunoglobulins, which was a cause of death in 2 patients. Adult patients with common variable immune deficiency received no adequate replacement therapy. Timely diagnosis and adequate therapy could not only preserve the life of many patients with severe PIDs (64.5% survived), but could achieve its relatively satisfactory quality. CONCLUSION: As of now, PIDs ceased to be fatal diseases. To improve the quality of health care to patients with this pathology, there is a need to increase the awareness of the diagnosis and treatment of immunodeficiencies among physicians of different specialties, to extend the application of molecular genetic techniques, including those for prenatal diagnosis, and to continuously provide patients with essential drugs.

[Oxidative stress, rRNA genes, and antioxidant enzymes in pathogenesis of schizophrenia and autism: modeling and clinical advices].

Ribosomal genes (RG), or genes for rRNA, are represented by multiple tandem repeats in eukaryotic genomes, and just a part of them is transcriptionally active. The quantity of active copies is a stable genome feature which determines the cell's capability for rapid synthesis of proteins, necessary to cope with stress conditions. Low number of active RG copies leads to reduced stress resistance and elevated risk of multifactorial disorders (MFD). Oxidative stress (OS) in the brain cells is believed to be involved in the pathogenesis of infantile autism (IA) and schizophrenia, i.e., MFDs with a manifested genetic predisposition. With autism, OS markers are found almost in every research, whilst with schizophrenia, the OS data are contradictory. Earlier, in a sample of patients with schizophrenia, we have found significantly higher quantity of active RG copies than at the average in healthy population. Here we have estimated the number of active RG copies in a sample of patients with IA (n = 51) and revealed significantly lower mean value than in healthy population. A novel mathematical model of the dynamic pattern of OS has been proposed. The model is realized as an ordinary differential equation system, supposing induction of antioxidant protection enzymes being mediated by reactive oxygen species (ROS), with the subsequent decrease of ROS content in a cell. The rate of synthesis of antioxidant protection enzymes is limited by the ribosome synthesis rate which depends on the number of active RG copies. Analysis of the model showed that the system always approaches a single stable equilibrium point along a damped oscillation trajectory, which in some degree resembles the dynamics of 'predator-prey' interaction in Lotka-Volterra model. The stationary ROS level inversely depends on the number of active RG copies. Our study explains the inconsistency of clinical data of OS in schizophrenia and suggests a novel criterion for discriminative cytogenetic diagnostics of schizophrenia and IA, as well as allows to assume that antioxidant therapy should be effective only for children with low number of active RG copies.

[Cluster size polymorphism of active human ribosomal genes and simulation of the conditions of its stability through generations].

Based on selective silver nitrate staining of active ribosomal gene (AcRG) clusters in nucleolus organizer regions (NORs) of human metaphase chromosomes, a technique was developed earlier to estimate the AcRG dosage in individual genomes as a sum of arbitrary units (0-3) ascribed to the silver precipitate (AgNOR) on ten NORs. The AcRG dosage was considered to be an additive quantitative trait determined by five polymorphic autosomal loci (with for allelic forms for each locus). A database was created to contain the data on AcRG cluster variants for more than 1000 individual human genomes. In this study, the population frequencies of AcRG cluster variants were determined. The results agreed with the hypothesis that stabilizing selection acts at the zygotic and/or early embryogenetic stage to restrain the AcRG genomic dosage (copy number) within a range from 14.9 to 23.7 arbitrary units (the cell is unviable when the trait is beyond this range). The average zygotic losses due to selection were estimated at 9.1-9.9% for a real population. A computer model where the AcRG dosage of a progeny results from a random combination of the AgNORs of the five acrocentric chromosome pairs of the parents was developed and used to simulate the formation of a certain AcRG genomic dosage through generations in a human panmictic population with nonoverlapping generations. A combination of stabilizing selection by total AcRG copy number and a certain spontaneous mutation rate (the probability of changes in the cluster size of a NOR as a result of unequal crossingover in meiotic prophase) was shown to be a sufficient condition for the restrain of equilibrium population frequencies of AgNOR size variants in a human panmictic population. Using the model, the most probable spontaneous mutation frequency was predicted to be (2.1-2.3) x 10(-2) per NOR per generation for human AgNORs. The predicted frequency was within the 95% confidence interval of the experimental rate, which was determined by studying the inheritance of AgNOR variants in real families.

[Determination of genome dose of active ribosome genes and several quantitative parameters of extracellular dna in test-subjects of the experiment with 7-day immersion].

Genome dose of active ribosome genes (ARG), average of nucleolus argyrophil structures in lymphocyte nuclei, levels of extracellular DNA (DNA(e)) concentrations and ratio of antibodies to total DNA (AB(DNA)) and ribosomal DNA (AB(DNA-rib)), and nuclease activity were determined in peripheral blood of 8 volunteered subjects (21-26 y.o.) in the experiment with 7-d DI. Results of the investigation revealed a broad individual variability ensued from heterogeneity of the group of the test-subjects as to ARG values. There was an inverse negative relationship between ARG values and increment of the ribosome genes activity index. Part of the subjected exhibited increased DNA(e) levels on completion of the experiment, whereas the others decreased the parameter demonstrating individual character of body reaction. No correlation was established between DNA(e) content and nuclease activity in blood. Concentrations of AB(DNA) and DNA AB(DNA-rib) before and after immersion were essentially unchanged; however, they were higher as compared with the control group of blood-donors. Diversity of subjects' reactions was accounted to the broad range of ARG values. Therefore, selection of test-subjects for ground-based simulation experiments should be conducted with due consideration of the parameter.

[Dynamics of blood biochemical parameters in an experiment with 7-day immersion].

Blood samples were taken from 8 volunteers (21 to 26 years of age) for 7-day immersion 7 days prior to, on days 3 and 7 of the experiment and on days 1 and 8 of recovery. Serum was analyzed for 38 biochemical markers of the functional state of the myocardium, skeletal muscles, hepatobiliary system, kidney, pancreas, GI tract, prostate, and protein-nucleic, carbohydrate, electrolyte and mineral metabolism. Seven-day immersion was found to alter the biochemical parameters within the physiological norm boundaries. The observed changes included lower activities of enzymes associated with muscular and myocardial constellation, shifts in electrolytes (K, Na, Mg), and increases in the biliary function parameters. Increased concentrations of the lipid metabolism parameters suggest a higher risk of atherogenesis. Biochemical parameters of bone tissues and erythrocyte activity were essentially unchanged. Most of the parameters returned to pre-experimental values by day 8 of recovery.

[The peculiarities of plantar pressure distribution during gait in different variants of functional system impairment in patients with multiple sclerosis].

Gait disturbance is a prominent disabling presentation of multiple sclerosis. The data on the distribution of plantar pressure during gait and its relation to the level of neurological deficit in various disturbances of pyramidal and cerebral functions in remitting type of multiple sclerosis are presented.

[Early and late responses to oxidative stress in human dermal fibroblasts of healthy donors and rheumatoid arthritis patients. Relationship between the cell death rate and the genomic dosage of active ribosomal genes].

A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.

[Effect of low-dose oral contraceptive regulon-Gedeon Richter on some hemostatic variables]. / Vliianie na nisko doziraniia oralen kontratseptiv regulon-Gedeon Richter vurkhu niakoi parametri na khemostazata

The authors have investigated the changes of some haemostatic variables at 33 women, who used low-dosed oral contraceptive REGULON--Gedeon Richter in the duration of six months. Statistically significant decrease is found in the activity of Protein S and Protein C, but plasminogen level is increased. The levels of the other variables remained in referential ranges. The use of REGULON doesn't influence the coagulation system.

[Telomerization as a method of obtaining immortal human cells preserving normal properties]. / Telomerizatsiia--sposob polycheniia immortal'nykh kletok cheloveka, sokhraniaiushchikh normal'nye svoistva.

Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.

[Hypoglycemic effect of an extract from Aronia melanocarpa leaves]. / Issledovanie gipoglikemicheskogo deistviia ékstrakta iz list'ev Aronia melanocarpa.

The action of extract from Aronia melanocarpa leaves to blood glucose level was investigated. It was shown that incorporated into drinking water and administrated intraperitoneally, the extract significantly reduce the blood glucose level of streptozotocin (STZ)-diabetic and normal rats.

New peptidomimetics of insulin.

New peptidomimetics that have been obtained in the course of our experimental work show distinct insulin-like activity both in vitro and in vivo. The first peptidomimetic (PM 1) is essentially a decapeptide in which sites of A (20-21) and B (19-26) chains of insulin are linked by the peptides bond (Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Cys-Asn). The second peptidomimetic (PM 2) has similar set of amino acid residues, except that two aromatic amino acids corresponding to the residues of B chain of insulin (B24 and B26) have been replaced with their D optical isomers (Cys-Gly-Glu-Arg-Gly-DPhe-Phe-DTyr-Cys-Asn). The third peptidomimetic (PM 3) has been obtained through acylation of N-terminal of PM 1 by the use of palmitic acid. The peptidomimetic incorporating D aromatic amino acids (PM 2) was demonstrated to exhibit more pronounced hypoglycemic impact, while the acylation of decapeptide tends to prolong the effective time of peptidomimetic influence in vivo.

[Stimulation of glucose uptake in PC 12 and L 929 cells by extracts from Aronia melanocarpa leaves]. / Stimuliatsiia pogloshcheniia gliukozy kletkami PC 12 i L 929 ékstraktom iz list'iev Aronia melanocarpa.

The ability of extract from Aronia melanocarpa leaves to increase glucose uptake was investigated. It was shown, that the extract stimulated glucose uptake by cells PC12 and L 929 at the concentration close to native insulin.

[The prolonged action of an insulin peptidomimetic by the substitution of L-amino acid for its D-isomer]. / Uvelichenie vremeni deistviia peptidomimetika insulina putem zameny L-aminokislotnykh ostatkov ikh D-opticheskimi izomerami.

The synthesized decapeptide represents functionally important site for binding to the insulin receptor. Amino acid residues at position, 1-8 correlate with B-chain of insulin at position B19-B26, and the residues at position 9-10 correlate with A-chain at position A20-A21. The new peptide was obtained by substitution of two aromatic L-amino acid residues (B24 and B26) for their D-optical isomers. These peptides were tested with cell cultures L929 and PC12 (glucose uptake). Increased concentration of peptides correlated with stimulation of glucose uptake by cells. Studies carried out on animals with streptosotocine-caused diabetes showed that, synthesized peptides were able to decrease glucose level in blood, but decapeptide with D amino acid showed a more pronounced effect compared to the decapeptide with L amino acid.