Vol 45, No 2 (2017)

Studies conducted in the recent years by biologists strongly suggest that physiological and reparative osteogenesis, as well as of the functional, adaptive, and post-traumatic reconstruction of bone tissues are based on common and stereotypical molecular and cellular mechanisms. Our experimental studies have shown that all stages of the bone microstructure morphogenesis are synchronously and continuously associated with focal and stereotypical angiogenesis (capillarogenesis). A powerful factor in the implementation of reparative osteogenesis is the osteoinductive interaction of the ends of the damaged bone segments, which positively shows itself even in cases of large diastasis between the fragments (provided that the fragments are steadily fixed). After any kind of stable osteosynthesis, by ensuring the stability of the bone fragments for the entire period of consolidation, an endosteal cortical bone regeneration by direct osteogenesis (i.e. without fibro-cartilaginous tissue) is observed in the minimum amount at the shortest time period. Periosteal bone formation in this case is actually a reserve source of bone formation, which becomes effective during insufficiently stable conditions. The instability of the bone damage area particularly that of the metal implants results in the most severe destructive consequences. 

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Background: The cause of late diagnosis of renal cancer lies in its durable, almost asymptomatic course. Due to the use of antiangiogenic therapies much progress has been made in its treatment in recent years. Yet, many questions concerning the diagnosis, prognosis and prediction of the efficiency of targeted therapy remain unsolved. Therefore, exploration of new renal cancer molecular markers, especially those related to its angiogenic and invasive activities, are still on the agenda. Such markers include the family of matrix metalloproteinases (MMPs) that degrade the majority of extracellular matrix components and are involved at all stages of tumor progression. Aim: Comparative evaluation of MMP2, 7, 8, 9 and type 1 tissue inhibitor (TIMP-1) levels in serum of healthy individuals and patients with renal cancer or benign renal tumors, analysis of their associations with the main clinical and pathologic characteristics of the disease. Materials and methods: We examined 99  renal cancer patients (of those 94 with primary tumor and 5 at progression) and 10 patients with benign renal tumors. The control group included 97 healthy individuals. Levels of the proteins studied were measured using respective direct ELISA kits (Quantikine^®, R&D Systems, USA). Results: MMP-7, MMP-8 and TIMP-1 levels in the sera of renal cancer patients were significantly higher than in the control group and in benign renal tumor patients. MMP-2 and MMP-9 levels did not differ significantly between the study and control groups. At MMP-7 cut-off level of 3.0  ng/mL, its diagnostic sensitivity for primary renal cancer was 84%, specificity in relation to “healthy” control – 87.5%, in relation to the pathologic control (healthy donors+benign renal tumor patients) – 73%. The best sensitivity: specificity ratio for TIMP-1 was 67 and 65% at the cut-off level of 315 ng/ml. No cut-off value with acceptable sensitivity: specificity ratio was found for MMP-8. Serum levels of all these 3 markers were positively associated with disease stage and TNM indices; MMP-7 and TIMP-1 levels also increased with lower differentiation grade. In 5 patients evaluated at disease progression the levels of all the markers studied were markedly higher than in the primary patients, and exceeded the estimated cut-off values. Conclusion: MMP-7 should be regarded as the most promising serological renal cancer marker; its serum levels exceed the cut-off value even in 84% stage I patients. TIMP-1 has acceptable sensitivity (70% and above) only from stage II renal cancer onwards, while MMP-8 levels are increased only at stage III–IV of the disease.

Rationale: Ovarian endometriosis is a progressive disease with growing prevalence and severity. Therefore, the development of robust non-invasive laboratory screening methods for early diagnosis on the out-patient basis seems quite relevant. Aim: To assess a potential of the quantitative phase imaging technique for early diagnosis of ovarian endometrial cysts and post-operative relapses of the disease. Materials and methods: We analyzed 1578 nuclei of the peripheral blood lymphocytes from 82 patients with ovarian endometrial cysts, aged 21 to 37 years (mean age 26.4 ± 3.6 years). The patients were follow-up in a gynecology out-patient clinic (the town of Yessentuki, Russia). Assessments were made longitudinally, i.e., before a laparoscopic cystectomy, at 6 and 12 months in the post-operative period with or without treatment with dienogest-containing agents. Morphological and functional status of the nuclei from the peripheral blood lymphocytes was assessed in the real-time mode by quantitative phase imaging (QPI) with the phase-interference microscopy module of the Bioni hardware and software complex (Westgrade Ltd., Moscow) for clinical and laboratory diagnostics, and the morphodensitometric segmentation technology. Results: The comparative analysis of morphometric parameters of CD3⁺ cells taken from peripheral blood of healthy non-pregnant women and patients with ovarian endometrial cysts before surgery showed a significant increase of the calculated functional activities of the lymphocyte nuclei (0.898 vs 0.783, p < 0.05). Assessment of changes overt time in the differential diagnostic criteria of the nuclear response in the peripheral blood lymphocytes from patients with endometrial ovarian cysts showed the following. Compared to the parameters obtained before treatment, at 6 and 12 months of the post-operative period the relative intensity of nuclear segments (ΔI) decreased by 10.3 and 14.7, 10.6 and 12.9% in the group treated with and without dienogest, respectively. Relative distance between the centers of the nuclear segments (ΔL) demonstrated a trend towards an increase by 0.6 and 0.9, 4.2 and 2.1%. The numbers of nuclear segments increased by 18.3 and 13.4, 27.4 and 16.9%, whereas the nuclear perimeter decreased by 13.9 and 12.6, 11.9 and 7.8%, respectively. In the patients treated with dienogest, the rate of non-relapse at 6 and 12 months of the follow-up was 100%, whereas in the patients without dienogest therapy, 97.5 and 93.5%, respectively. Discussion: Interphase chromatin is a unique biosensor of the early abnormalities in a lymphoid cell. Modification of its structure and packaging density not only indicate changes of the morphofunctional status of the lymphocyte, but can be projected to the body as a whole and used for early pre-clinical diagnosis, assessment of severity of the pathological process and prediction of the outcome in various critic states. Conclusion: Practical implementation of QPI for clinical monitoring of patients with ovarian endometrial cysts makes it possible to obtain important information on the cell immunity in real time. It opens new opportunities to assess the efficacy of treatment and rehabilitation activities, as well as for early pre-clinical diagnosis of relapsing disease.

Rationale: A  significant proportion of post-operative infectious complications in cardiovascular surgery is caused by Staphylococcus spp, with an increasing role of Staphylococcus epidermidis. Aim: To identify biological characteristics of coagulase-negative staphylococci isolated from various biotopes in a cardiac surgery department, in order to assess their pathogenic potential. Materials and methods: The study was performed with Staphylococcus isolates (n = 73) obtained in a cardiac surgery department in 2015–2016. Biochemical identification of the cultures was done with the use of Vitek 2  compact microbiological analyzer (BioMerieux, France) and test systems Staphytest (ERBA Lachema, Czech Republic). The biological properties of staphylococci were assessed by classical microbiological techniques. Statistical analysis was done with the Microbe-2  software package. Results: S. aureus and 6 strains of coagulase-negative staphylococci with predominance of S.  epidermidis were isolated in the department of cardiac surgery. In most cases S.  epidermidis was isolated from post-operative wounds (n = 16, 30.8 ± 6.4%) and environmental swabs (n = 11, 21.1 ± 5.7%). All S.  epidermidis studied (n = 52) hydrolyzed maltose with formation of the acid without gas, could ferment glucose and tregalose, and were lacking β-galactosidase (except the cultures isolated from post-operative wounds). They also could not degrade mannitol in aerobic environment (excluding staphylococci isolated from the environmental objects and from the medical staff). S.  epidermidis displayed variable biochemical activity towards lactose and saccharose. Hemolytic activity (more often of the β-type) was characteristic for 69.2 ± 6.4%  of S.  epidermidis cultures. The majority of cultures (92.4 ± 3.6%) displayed proteolytic activity irrespective of the type of staphylococci and the source of isolation. Lipolytic activity was more frequent in staphylococci isolated from clinical materials (airway secretions of the medical personnel [9/10  cultures], patients [9/10  cultures] and post-operative wounds [16/20 cultures]), than in those isolated from the environmental objects. There was a direct significant correlation between the lipase activity and the presence of β-galactosidase (φ=0.40), lipase and proteolytic activities (φ=0.33), β-galactosidase and gelatin liquation (φ=0,65), hemolytic and proteolytic activities (φ=0.37). Conclusion: S. epidermidis was the most prevalent pathogen in all ecotopes of the department of cardiac surgery. Biochemical properties of coagulase-negative staphylococci and their fermentative activity related to factors of pathogenicity were different depending on the site of isolation. This illustrates their contribution in the development of inflammation in a given biotope.

Rationale: Enterococci are the leading cause of a number of nosocomial and community-acquired human diseases. In the last decade, these pathogens are becoming resistant to antibacterials, including vancomycin. Multidrug-resistant enterococci have been also isolated from agricultural animals in many countries worldwide, which raises concern of scientists because of possible horizontal transfer of resistance genes. Aim: To assess antibacterial sensitivity of Enterococcus spp. isolates collected from the poultry in the Russian Federation from 2013 to 2016, and to identify vancomycin-resistance genes in their genomes. Materials and methods: Eighty-seven enterococci isolates belonging to E.  faecalis (n = 47, 54%), E.  faecium (n = 25, 28.7%) and other species (n = 15, 17.2%) were collected from clinical samples of 297 heads of poultry (liver, lungs, heart, spleen, contents of the nasal and sinus cavities) from 17  poultry farms of the Northwest, Central, Volga, Ural and Southern Federal districts of the Russian Federation. Sensitivity of enterococci to antibacterials was determined by disk-diffusion and broth microdilution methods. Vancomycin resistance genes van was detected by polymerase chain reaction with specific primers. Results: Most enterococci isolates were resistant to erythromycin (74/87, 85.1%), gentamicin (70/87, 80.5%), ceftriaxone (61/87, 70.1%), ciprofloxacin (56/87, 64.4%), tetracycline (57/87, 65.5%), and rifampicin (48/87, 55.2%), fewer ones to trimethoprim (38/87, 43.7%), ampicillin (28/87, 32.2%), linezolid (15/87, 17.2%) and chloramphenicol (5/87, 5.7%). The vanC type genes (vanC1 and vanC2/3) were identified in 10  isolates. Vancomycin minimal inhibitory concentrations for these isolates were 2 to 8  mg/L. E. faecium with vanC1 gene was isolated from poultry probably for the first time ever. Conclusion: Commercial poultry in the Russian poultry farms is an important reservoir and source of antibiotic-resistant enterococci populations, including enterococci carrying vanC1 and vanC2/3 vancomycin resistance genes. 

Background: Adhesion and ability to form a  biofilm are considered among the leading pathogenicity factors of Corynebacterium diphtheriaе, responsible for bacterial carriage. It is exactly bacterial carriage that ensures the circulation of diphtheria pathogen strains in the inter-epidemic periods. Aim: To assess and compare adhesivity of standard and biofilm cultures of toxigenic C. diphtheriae strains. Materials and methods: We studied standard and biofilm (120  and 720  hour) cultures of C.  diphtheriae strains. Their ability to form a  biofilm was tested according to P. Watnick (2000). Adhesivity was assessed in the pharyngeal epithelial carcinoma Hep-2 cell culture with various time exposures (2, 8, and 18  hours). The amounts of C. diphtheriae adhered to Нер-2 cells were measured by culturing the swabs in the 20% serum agar with subsequent calculation of mean numbers of colony-forming units (CFU) per 1  mL. Results: All standard and biofilm cultures of the studied toxigenic strains of C. diphtheriae had adhesive properties of various degrees. The highest adhesivity was found in a  circulating strain C.  diphtheriae gravis tox+ (from 0.26 ± 0.01 to 203.3 ± 3.3 CFU/mL), which differed from the same parameters in other strains studied (from 0.03 ± 0.003 to 0.20 ± 0.01  CFU/mL). The lowest adhesivity after a 2-hour exposure was found both in the standard and biofilm cultures of C.  diphtheriae gravis tox+ 6765, whereas after the exposure of 8 and 18  hours, the lowest adhesion properties were demonstrated by C.  diphtheriae gravis with a “silent” tox gene and C. diphtheriae mitis tox+ 269. All cultures of toxigenic C. diphtheriae strains showed a  statistically significant increase in their adhesivity (р ≤ 0.05) by 8 and 18  hour of cultivation. Conclusion: Circulating C.  diphtheriae gravis tox+ strain demonstrated the highest adhesivity among all toxigenic strains of the diphtheria pathogens studied. 

Background: Quantitative luminescent analysis is widely used in biology, laboratory diagnostics and clinical medicine to study objects at various levels. However, the existing simplified algorithms for calculation of the luminophore concentration in diluted linear solutions cannot be applied to the turbid media with strong light scattering, which include the majority of living biological tissues. Today, the development of luminescence analysis in medicine goes towards the creation of non-destructive and non-invasive methods of in vivo monitoring. Therefore, the urgent question is about a formulation of the research purpose and the development of algorithms to compute the luminophore concentration based on the luminescence spectra recorded in the turbid media. Objective: To formulate and justify the tasks of elaboration of the algorithms to compute the luminophore concentration based on the luminescence spectra recorded under conditions of the optically turbid media. Materials and methods: We looked upon the physico-mathematical simulation of the process of formation of the induced fluorescence emission in the light-scattering medium based on the modified 2-flux Kubelka-Munch model. We conducted a series of laboratory experiments with macro-homogenous light-scattering model media to determine characteristics of the dependence of the fluorescence intensity detected from the surface of an optically turbid biological medium upon the factor of light scattering and the concentration of the fluorophore in the medium. Results: Both theoretical and experimental results demonstrate complex nonlinear dependence of the fluorescence intensity detected upon the optical properties and a  concentration of a  fluorophore in the turbid media. This dependence is very different from the known linear  C.  Parker's solution for transparent media, which makes it impossible to use it in the optically turbid media. Conclusion: Further studies searching a  closed-form analytical solution of the inverse optical problem for light-scattering and fluorescent media are necessary to calculate the luminophore concentration in a light-scattering media based on the recorded luminescence spectra.