Vol 46, No 8 (2018)

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.

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.

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.

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.

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.