Vol 46, No 8 (2018)


Expression levels of the apoptosis genes FAS, TNFR2, TRAIL, DR3 and DR4/5 in patients with newly diagnosed chronic lymphatic leukemia before and after treatment with fludarabine, cyclophosphamide and rituximab (FCR)

Zakharov S.G., Golenkov A.K., Misyurin V.A., Kataeva E.V., Baryshnikova M.A., Chuksina Y.Y., Mitina T.A., Trifonova E.V., Vysotskaya L.L., Chernykh Y.B., Klinushkina E.F., Belousov K.A., Finashutina Y.P., Misyurin A.V.


Background: We have previously shown that the FAS, TNFR2, TRAIL, DR3, DR4/5 gene expression in patients with newly diagnosed chronic lymphoblastic leukemia (CLL) correlates with clinical manifestations of the disease: they are minimal in patients with high activity of the proapoptotic genes and low activity of the apoptosis-inhibiting genes, and advanced in patients with high expression of the anti-apoptotic and low expression of the pro-apoptotic genes.

Aim: To compare the levels of expression of the external apoptosis pathway genes in patients with newly diagnosed CLL before and after chemotherapy with fludarabine, cyclophosphamide and rituximab (FCR), taking into account baseline clinical data and the response to treatment.

Materials and methods: This prospective one-center cohort study included 23 patients with newly diagnosed CLL, who underwent clinical and diagnostic assessments and treatment from November 2014 to December 2017. Immunophenotyping of peripheral blood lymphocytes for CLL diagnosis was done by fourcolor flow cytometry. Expression of the external apoptosis pathway genes was assessed by realtime reverse transcriptase polymerase chain reaction. All patients were treated with a standard FCR regimen with subsequent maintenance treatment with rituximab.

Results: There were more men (n = 16) than women among our 23 CLL patients. Median age was 64 years (range, from 47 to 77 years). Sixteen (16) patients had CLL Rai Grade I and II, and 7 patients had CLL Grades III and IV. For convenience of analysis, all patients were divided into two groups depending on the FAS gene expression. At baseline, the patients with high FAS expression had higher TNFR2 (p < 0.0015) and TRAIL (p < 0.0053) expression levels. Before FCR therapy, the patients with low FAS expression had higher lymphocyte counts (р = 0.0016) and lower erythrocyte counts (р = 0.0159). At baseline, there were more Grade I and II patients in the group with higher FAS expression (р = 0.0205). At day 3 after the end of a four day FCR cycle, there was an increase only of the FAS (p = 0.0025) and TRAIL (p = 0.0045) expression. After the completion of the first FCR cycle, lymphocyte counts in the patients with low FAS expression decreased earlier than those in the patients with high FAS expression (p = 0.0019). After six FCR cycles, complete or partial remission was obtained in 82% (19/23) of the patients. The patients with high FAS expression had higher complete remission rate (р = 0.026). No adverse events related to FCR were registered.

Conclusion: The external apoptosis pathway genes are one of the key factors of the tumor progression in CLL. Our data on the effect of FCR therapy on the FAS and TRAIL gene expression make it possible to consider them as a target for this combination regimen and may become the rationale to develop new pharmaceutical molecules.

Almanac of Clinical Medicine. 2018;46(8):734-741
pages 734-741 views

Evaluation of nonlinear dynamics of the cell structure damage as a promising method for personalized cancer diagnostics

Naimark O.B., Nikityuk A.S., Nebogatikov V.O., Grishko V.V.


Background: Modulation interference microscopy is one of the promising technologies for early personalized cancer diagnostics, while it gives the possibility to obtain real-time images of living functioning cells at a nanometer resolution, as well as to assess spatio-temporal changes of their intracellular structures.

Aim: To identify the markers of malignant transformation of cells based on a multiple scale spatio-temporal of the data obtained by lase interferometry during in situ registration of changes in intracellular structures.

Materials and methods: The study was performed with the breast adenocarcinoma MCF-7 cell line and human kidney epithelial cells HEK 293 provided by the N.N. Blokhin National Medical Research Centre of Oncology (Moscow, Russia). Changes in fluctuation of the living cell phase thickness were measured with a laser modulation interference microscope MIM-340 (PA UOMZ, Russia). The multifractal detrended fluctuation analysis (MF-DFA) was used to assess the results obtained.

Results: By comparison of the scaling exponential spectra and corresponding spectra of the singular fluctuations in the minimal and maximal diameters of the phase pictures of HEK 293 cells and MCF-7 cells, it was shown that the cancer cell dynamics has definite monofractal properties: a linear dependency of the scaling exponents τ from the actual q parameter and “dotted” singularity spectrum (the spectral width does not exceed 0.1). The dynamics of healthy cell demonstrated multifractal properties, such as a non-linear dependency τ(q) and a bell shaped singularity spectrum (the spectral width over 0.1).

Conclusion: We have proposed a new approach to assess the intracellular structural damage during oncogenesis. The signs of monofractality are objective criteria of malignization. The results obtained seems promising as early diagnostic markers of cancer transformation (damage) of cells and determination of tumor cell sensitivity / resistance to anti-tumor agents in an individual patient.

Almanac of Clinical Medicine. 2018;46(8):742-747
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A dialogue with the cell: diagnostic real-time technology based on laser interferometry

Vasilenko I.A., Metelin V.B., Ignat'ev P.S., Kardashova Z.Z., Lifenko R.A.


The paper presents a review of current techniques of the interference microscopy of living functioning cells that allow for assessment of the real-time changes of subcellular microstructures with 2D and 3D reconstructions of the images and multifactorial data analysis. It has been shown that new diagnostic information can be obtained within the research methodology of the so-called interactive dialogue with the cell to identify changes in the morphofunctional state of living cells through registration of their realtime response to environmental fluctuations. We describe physical basis of laser interferometry, problems and specifics of interpretation of cell phase pictures. As an illustration to the practical use of the technique in the clinic we present the results of studies on morphofunctional state of peripheral blood platelets in patients with oral malignancies (OM) and various stages of the tumor. Analysis of circulating platelets with a realtime technique of laser interferometry allowed for identification of compensated activation of the platelet compartment of hemostasis in these patients, as well as an association between the severity of cancer and changes of dimensional cell parameters (an increase of the mean diameter and area of circulating platelets by 23.4, 26.8 and 30.3%, and by 30.2, 32.5 and 38.1% in the patients with OM grades II, III and IV, respectively (p < 0,05 for all comparisons)). The necessary condition for a wider implementation of new diagnostic technologies is a critical evaluation of the efficacy of their use in routine practice, identification of their advantages, disadvantages and differences between current and proposed diagnostic platforms.

Almanac of Clinical Medicine. 2018;46(8):748-757
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The potential and prospects of the quantitative phase imaging (QPI) diagnostic technology in obstetrics and gynecology

Lifenko R.A., Popova O.S., Topuzov A.G., Zverzhhovsky V.D.


Rationale: An important role of immunological factors in the endometriosis pathophysiology allows for their consideration as diagnostic and prognostic disease markers. A promising approach to the diagnosis of immunological abnormalities is based on the non-invasive cell diagnostic technology of quantitative phase imaging (QPI), with underlying principles of laser interferometry and holography.

Aim: To assess the QPI potential to identify diagnostic criteria for cell immunity abnormalities in female patients with external endometriosis.

Materials and methods: We performed a quantitative assessment of the cytotoxic potential of peripheral blood lymphocytes from 22 patients with endometrioid ovarian cysts (mean age, 27.1 ± 3.4 years), 20 patients with uterine leiomyoma (mean age, 29.3 ± 4.1 years) and 20 health non-pregnant women of child-bearing age (mean age, 28.7 ± 3.6 years). We used the non-invasive QPI technique with the module for phase interference microscopy of the Russian hardand software complex Bioni (Westtrade Ltd., Russia).

Results: We studied phase interference pictures of native lymphocytes to identify morphological and densitometry characteristics for CD4+ и CD8+ cells. Two groups of cells were identified in the cytotoxic lymphocyte population, which differed in their values of the phase thickness (Hcyt) and the area of cytoplasm adjacent to the membrane and containing the perforin granules (Scyt). It was found, that in the healthy controls the proportion of perforin-positive cytotoxic CD8+ lymphocytes was 37.1 ± 5.15%, in the patients with endometriosis 29.8 ± 6.34%, and in those with uterine leiomyoma 42.6 ± 5.89%.

Discussion: Assessment of the lymphocyte cytotoxic potential by quantitative determination of cells containing perforin granules may be an important diagnostic and prognostic criterion of the disease course.

Conclusion: Studies of the intracellular structures of lymphocytes by non-invasive and non-reagent technology QPI allows not only for a detailed evaluation of pathophysiological mechanisms of immune competent cell transformation in the course of a pathological process, but also gives future directions for development of new approaches to functional assessment of the immune system based on the results obtained.

Almanac of Clinical Medicine. 2018;46(8):758-764
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Translational studies of electrophoretic mobility and phase picture of erythrocytes with consideration of development of stress response during a pathological process

Deryugina A.V., Ivashchenko M.N., Ignat'ev P.S., Samodelkin A.G.


Rationale: Modern cell diagnostic methods are in high demand during the development of new approaches in personalized medicine. Coherent phase interferometry and cell microelectrophoresis are among such methods that are being actively introduced into the diagnostic process in medical institutions.

Aim: To substantiate the potential use of biophysical and morphodensitometrical erythrocytes parameters as criteria of treatment efcacy and course of adaptation process in patients with gastrointestinal tract disorders.

Materials and methods: The study included 25 patients aged from 40 to 54 years (11 males and 14 females), among them 9 (36%) with gastric peptic ulcer, 3 (12%) with duodenal ulcer, 8 (32%) with acute gastritis, and 5 (20%) with acute pancreatitis. Biophysical and morphological particulars of peripheral blood erythrocytes were assessed before and after treatment using cell diagnostic techniques, such as microelectrophoresis and laser modulation interference microscopy. Also, we evaluated changes over time in routine clinical laboratory tests, such as red and white blood cell counts, hemoglobin levels, and erythrocyte sedimentation rate (ESR), and differential leukocyte counts. The control group included 10 healthy donors aged from 36 to 52 years. In vitro experiments were performed to assess the erythrocyte electrophoretic mobility (EEPM) and morphology of erythrocytes treated with epinephrine or cortisol.

Results: After the treatment, the patients demonstrated a decrease in their leukocyte counts (by 27%), a 2-fold increase in monocyte counts and an ESR decrease (by 10%), compared to the corresponding baseline values before treatment (p < 0.05 for all comparisons). EEPM increased by 12% (1.37 vs. 1.22 mcm × cm/V × s, p < 0.05). The erythrocyte pool of the patients before treatment, had a decreased proportion of discocytes, compared to that in the control group (85.2 vs. 95.4%, р < 0.05), increased proportions of echinocytes, stomatocytes and degenerative forms (11, 2.8 and 1%, respectively, р < 0.05). After the treatment, the discocytes counts increased virtually up to their physiological normal range (91.3%). However, the surface of the discoid cells remained heterogeneous with multiple microspicules; this resulted in changes of electrokinetic and morphological properties of erythrocyte response to stress reaction occurring in the body. The impact of the stress effectors was confrmed in in vitro experiments assessing the effects of epinephrine (1 × 10-9 g/mL) and cortisol (5 × 10-7 g/mL) on erythrocytes. At 120 minutes of the experiment, epinephrine decreased EEPM (1.14 vs. 1.24 mcm × cm/V × s at baseline, р < 0.05) and increased cell sphericity. On the contrary, cortisol increased EEPM (1.72 vs. 1.36 mcm × cm/V × s, р < 0.05), with non-signifcant echinocytic transformation.

Conclusion: Biophysical and morphodensitometric parameters of red blood cells obtained with the use of current express methods of cell microelectrophoresis and coherent interference microscopy help to objectivize the intensity of stress response during a pathological process and activation of adaptation mechanisms during the treatment.

Almanac of Clinical Medicine. 2018;46(8):765-771
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Changes in plasma spectral characteristics during the in vitro contact of human venous blood with granulated sorbents

Burkova N.V., Kirichuk O.P., Romanchuk E.V., Davankov V.A., Postnov V.N., Kuznetsov S.I.


Rationale: During hemosorption procedures, it is important to investigate not only the sorption and activation characteristics of hemocontact agents, but also to assess the effect of sorbents on the parameters of blood homeostasis. The intensity of hemolysis can be judged by the degree of changes in optical density of blood plasma at wavelengths corresponding to the peak absorption of hemoglobin (414, 544 and 577 nm).

Aim: To assess the effect of three granular sorbents (SKT-6A, HPS, Silochrome C-120) contacting human venous blood in vitro on changes in plasma spectral characteristics.

Materials and methods: The blood contact was modeled at bench conditions with the use of donated blood at rotating mode. Blood samples were drawn before the experiment and after 5, 20, 40, and 60 minutes. Spectroscopic assessment was performed in the visible light range (300–700 nm) with UNICO 2802(S) spectrophotometer.

Results: The interaction of the SKT-6A sorbent with blood resulted in a 17.3% decrease in the plasma optical density at a wavelength of 540 nm, compared to baseline, as soon as at 5 minute of the experiment (p < 0.05). The decline in optical density imposed by the blood contact with HPS ranged from 2.6 to 12.1% (p < 0.05) during the observation period. The sorption activity of SKT-6A and HPS prevailed over their lytic properties. On the contrary, the percentage change in the optical density of the Silochrome C-120 sorbent during its blood contact increased from 25.6 to 38.3% (p < 0.05), indicating that this sorbent was inducing hemolysis. The sorbents tested can be arranged as follows according to their ability to induce hemolysis during their contact with blood: HPS < SKT-6A < Silochrome C-120.

Conclusion: The tested SKT-6A and HPS sorbents can be used as blood-contact agents for the low volume hemoperfusion. The HPS agent seems to be the most promising for routine clinical use. The Silochrome C-120 sorbent requires some chemical modification to improve its properties of hemocompatibility.

Almanac of Clinical Medicine. 2018;46(8):772-777
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The use of the interference microscopy to study structural characteristics of cultured dermal fibroblasts

Nefedova I.F., Rossinskaya V.V.


Rationale: The use of electron, nuclear power and confocal microscopy for the screening of biologically active compounds, medical products and express diagnostics of some diseases at the cell level is associated with laborand time-consuming sample preparation, which cannot exclude potential measurement errors and artifacts. The modulation interference microscopy does not have these disadvantages; it allows for non-invasive studies of cell structures, imaging with nanometer resolution and analysis of the optical properties of an object.

Aim: To assess the potential of the interference microscopy in the evaluation of morphofunctional characteristics of in vitro mitomycin conditioned cultured cell nuclei (dermal fibroblasts taken as a model).

Materials and methods: Native culture of human dermal fibroblasts of the 6th passage, grown on glass with mirror coating in the cell culture laboratory of the Institute of Experimental Medicine and Biotechnology of Samara State Medical University (Russia), was examined with a modulation interference microscope MIM-340 (JSC PA UOMZ, Russia). Changes over time in the structural characteristics of dermal fbroblast nuclei conditioned with mitomycin were evaluated. The control group included fibroblasts cultured in the same conditions on glass with mirror coating without mitomycin. Imaging with MIM-340 was done at three hours, one and four days after adding the cytostatic. The control group was assessed at the same time points.

Results: We have shown that the cell culture grown on dielectric glasses does not differ in its morphofunctional characteristics from the culture grown on culture plastics. This proves the possibility to study the adhesive native culture using interference microscopy. We have found that the cells respond to a single mitomycin 0.04% exposure with a change to a globular shape and a sharp increase in the nuclear phase thickness (217.8 vs. 142.18 nm in the control group, p ≤ 0.05). Thereafter, the morphofunctional characteristics of the cells are restored, which is confirmed by the changes over time in the culture density, cell shape and size, and the phase thickness of the nucleus.

Conclusion: The results obtained make it possible to recommend the method of modulation interference microscopy for evaluation of toxicity and biocompatibility of drugs, medical products and physical factors for diagnosis and treatment.

Almanac of Clinical Medicine. 2018;46(8):778-783
pages 778-783 views

Age-related changes in production of short chain fatty acids by oropharyngeal microbiota in patients without respiratory tract and oral disorders

Zatevalov A.M., Selkova E.P., Gudova N.V., Oganesyan A.S.


Rationale: Functional activity of upper respiratory tract and oral microbiota has a high informational potential for diagnostics of infectious disease and for development of preventive measure, which is to be explained by rapid variability and high activity of bacteria in this location.

Aim: To determine statistical characteristics of concentrations and ratios of the oropharyngeal short chain fatty acids (SCFA) (i.e., functional activity of oropharyngeal microbiota) depending on age of patients without infectious disorder of upper respiratory tract and oral cavity.

Materials and methods: Gas liquid chromatography was used to measure SCFA concentrations in saliva from 683 patients aged from 1 month to 85 years who did not have any infections of respiratory tract and oral cavity. Age intervals with homogenous salivary SCFA levels were determined with constant trends in their means (medians) with one-month accuracy. The resulting parameters for the identified age intervals were compared with Mann-Whitney test at 95% significance level (p < 0.05).

Results: There were no significant differences between median total SCFA levels (8.04 [4.85; 14.22] mmol/G) and median acetic acid levels (6.27 [3.79; 11.21] mmol/G) in saliva from patients of all ages from 1 month to 85 years. For all other parameters, from 2 to 3 steps of changes were found that occurred at the age of 4 months and 14 years. After the age of 14, the concentrations of propionic and butyric acid significantly increased, whereas those of valeric and caproic acids, as well as of the branched chain SCFA decreased. Correspondingly, after the age of 14, the mean structural index increased from 0.25 to 0.27 U (p < 0.05). The isoacid index decreased with age in two steps: at the age of 4 months from 1.89 to 1.04 U (p < 0.05) and later at the age of 14 years to 0.74 U (p < 0.05).

Conclusion: Salivary SCFA levels become stable at the age of 14. The structural index and the isoacid index are most sensitive to the integral changes in the microbiota composition. Analysis of the results of studies on metabolic functioning of microflora should be based on mathematic modeling and multifactorial statistics in three age intervals: from birth to 4 months of age, from 4 months to 14 years and over 14 years.
Almanac of Clinical Medicine. 2018;46(8):784-791
pages 784-791 views

Plasma catecholamine levels in the early stages of treatment-naïve Parkinson’s disease

Zalyalova Z.A., Khasanova D.M., Ugrumov M.V.


Rationale: Parkinson's disease (PD) is a neurodegenerative disorder with predominant involvement of catecholamine-producing neurons of the central and peripheral nervous system. Taking into account the relative availability and low costs of plasma catecholamine measurements, it is worthwhile to study these parameters as biomarkers of the early stages of PD.

Aim: To determinate whether plasma levels of dopamine (DA), norepinephrine (NE), L-3,4-dihydroxyphenylalanine (DOPA) and dihydroxyphenylacetic acid (DOPAC) in patients with early stages of PD are related with akinetic-rigid and tremor-dominant variants and to compare the results to healthy volunteers.

Materials and methods: This was an observational cross-sectional cohort study performed from 2012 to 2015. The main study group included unselected outpatients who attended the Republican Consultative and Diagnostic Center of Movement Disorders and Botulinotherapy (Kazan, Russia) with newly diagnosed early PD (Hoehn and Yahr stages I and II, 1967), of various ages and both genders, who had not been given any specific antiparkinsonian treatment. The control group included healthy volunteers with no clinical signs of PD (they could have other chronic diseases of the non-extrapyramidal origin). Plasma catecholamine levels were measured by gas liquid chromatography.

Results: One hundred and thirty (130) treatment-naïve patients with newly diagnosed PD (mean age 59.34 ± 8.42 years, male gender 45.38%) were enrolled into the main study group. The control group included 56 healthy volunteers matched for age and gender. The distribution of various PD forms and stages was as follows: PD tremor-dominant variant 56.9%, PD akinetic-rigid variant 43.1%; PD stage I 76.9%, PD stage II 23.1%. Irrespective of the variant and stage, the PD patients demonstrated decreased NE levels, compared to the controls (95% confidence intervals 124–216 and 248–428 pg/mL, respectively, р < 0.026). DOPA plasma level was reduced only in the patients with akinetic-rigid PD variant (р = 0.017), while DOPAC level in the patients with PD stage II (р = 0.008). The average DA:NE:DOPA:DOPAC ratio was 1:32:105:64 in the control group, 1:62:238:88 in the patients with PD tremor-dominant variant (the difference is significant for NE and DOPA, р < 0.05), and 1:29:96:32 in those with PD akinetic-rigid variant (p > 0.05). In the healthy controls the changes in DOPA levels account for 84% of the DA and NE variability; no correlation between DOPAC and other catecholamines was found. On the contrary, in the PD patients regardless of the stage and the disease variant, DOPAC levels directly correlated with DA (p < 0.04). The PD tremor-dominant variant patients demonstrated a direct correlation between plasma NE and DOPA levels (p < 0.05).

Conclusion: The results obtained on absolute and relative parameters catecholamine turnover in the patients with early PD stages support the hypothesis on different pathophysiology of the tremor-dominant and akinetic-rigid variants of PD.

Almanac of Clinical Medicine. 2018;46(8):792-801
pages 792-801 views

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