Endogenous Regeneration of Alveolar Bone by Decellularized Tooth Matrix.

The efficiency of bone tissue regeneration by decellularized tooth matrix, demineralized tooth matrix, and commercial xenograft Bio-Oss Spongiosa was compared on the model of a critical-size circular defect in the alveolar bone of the upper jaw of adult Wistar rats. The defect healing dynamics was assessed using histological, histomorphometrical, and immunohistochemical methods on days 30 and 60. In contrast to demineralized matrix and commercial xenograft, decellularized matrix induces the formation of the new bone tissue by day 60. Decellularized matrix can be considered as a biomaterial for cell-free tissue engineering for alveolar bone restoration in dentistry and maxillofacial surgery.

Molecular Genetic Characterization of Streptococcus pyogenes Strains Isolated from Patients with Various Manifestations of Streptococcal Infection.

In 82 clinical strains of Streptococcus pyogenes (group A streptococci) isolated from patients with various manifestations of streptococcal infection, emm-typing revealed 27 emm-types (n=77) with a predominance of emm-89 (n=15; 18%), emm-75 (n=9; 11%), and emm-1 (n=6; 7%); types emm-3, emm-12, and emm-58 (n=4; 5% each) were found with almost equal frequency; other types were less common. The superantigen genes speC, speG, speH, speI, speJ, speK, speL, speM, smeZ, and SSA were identified in S. pyogenes strains using multiprimer PCR; the genes of the superantigen SpeA and cysteine proteinase SpeB were detected using real-time PCR. All the studied S. pyogenes strains contained superantigen genes, and 98% of the strains had several (from 2 to 7) genes. The number of variants of these sets reached 37; 2% of the strains contained only one superantigen gene. The distribution frequencies of superantigen genes in the studied strains were: speA - 43%; speC - 38%; speG - 93%; speH - 13%; speI - 6%; speJ - 24%; speK - 13%; speL and speM - 11% each; smeZ - 98%; SSA - 15%. All studied S. pyogenes strains contained the speB gene. Our studies have demonstrated that the sets of superantigen genes of group A streptococci are characterized by pronounced diversity to some extent associated with emm-type.

The Inhibitory Activity of Lactobacillus plantarum Supernatant against Enterobacteria, Campylobacter, and Tumor Cells.

Cell-free supernatant of Lactobacillus plantarum exhibit a strong antimicrobial effect against a number of pathogenic enterobacteria (E. coli, Shigella flexneri, Salmonella typhimurium, Proteus mirabilis, and Campylobacter jejuni). The degree of growth inhibition in broth culture reached a high level for all tested bacteria. The highest rates were noted for P. mirabilis (by 13 times) and the lowest for S. flexneri (by 5 times) and C. jejuni (by 4.5 times). Significant antiproliferative effect of the supernatant on cells of tumor-derived epithelial cell lines was shown. The highest degree of inhibition (by 22 times) was observed for HT-29 cells (colon carcinoma). Thus, inclusion of probiotics in traditional treatment schemes can increase the effectiveness of antibacterial and antitumor drug therapy.

Antibacterial Activity of Lactobacillus plantarum Supernatant on Non-Fermenting Gram-Negative Bacteria.

We studied the effect of the L. plantarum strain supernatant on the growth of culture and biofilm of non-fermenting bacteria of the genera Pseudomonas, Achromobacter, and Burkholderia. To obtain a supernatant, the culture of L. plantarum was grown for 48 h at 37°C on a Lactic broth nutrient medium with casein peptone, then centrifuged and filtered through a 0.22-µm Millipore filter. Antimicrobial activity was determined by broth microdilution assay. The inhibitory effect of the supernatant on the growth of bacteria of all three genera was demonstrated. The maximum inhibition was observed for P. aeruginosa (by 13 times compared to the control). For bacteria of the Achromobacter and Burkholderia genera, the inhibition was less pronounced: by 7 and 6 times, respectively. The supernatant also inhibited biofilm formation by P. aeruginosa and A. ruhlandii, but did not affect formed biofilm. Thus, the L. plantarum supernatant obtained by us exhibited pronounced antimicrobial activity against non-fermenting bacteria, the causative agents of nosocomial infections, especially in immunocompromised individuals, very often in cystic fibrosis patients.

Inhibitory Effect of Copper and Zinc Ions on the Growth of Streptococcus pyogenes and Escherichia coli Biofilms.

Analysis of the effect of copper and zinc ions on Streptococcus pyogenes and Escherichia coli biofilms revealed significant differences in the effect of these metals in the form of sulfates or chlorides on biofilm formation. Zinc ions in low doses (salt concentration 0.005 M) inhibited the growth of S. pyogenes biofilms by 1.5 times. After increasing salt concentration to 0.05-0.5 M, the growth of biofilm was reduced by 2.5 times in comparison with the positive control. In case of E. coli biofilms, the inhibition was more pronounced: zinc sulfate in a concentration of 0.005 M reduced its growth by 4.6 times in comparison with the positive control. After increasing salt concentration, the growth of E. coli biofilm decreased by 6.8 times. In case of zinc chloride, zinc ions produced weaker effect and reduced biofilm growth by 2.2 and 5 times, respectively. Copper salts in a concentration of 0.005 M had practically no effect on the growth of S. pyogenes biofilm; with increasing salt concentration, the degree of inhibition was close to the effect of zinc. In case of E. coli biofilm, we observed a slight inhibition of the growth by low doses of copper ions (by 1.4-1.3 times); with increasing salt concentration the effect increased by 5.6 and 2.2 times for copper sulfate and chloride, respectively. Copper and zinc cations had no effect on mature biofilm.

Osteogenic Potential of Decellularized Tooth Matrix.

We studied the possibility of using decellularized tooth matrix as a scaffold to restore bone tissue. It was found that mesenchymal stem cells underwent spontaneous osteogenic differentiation on the decellularized tooth matrix, which makes it possible to use it as a natural allograft in the treatment of resorption of alveolar bone tissue.

Peculiarities of Osteogenesis by Periosteal Cells after Experimental Ectopic Transplantation.

We carried out a comparative study of the features of osteogenesis from the progenitor osteogenic periosteal cells in rabbit and human. At the initial stages, high osteogenic potential of both human and rabbit periosteal cells was observed. However, at the later stages, the cell response favors resorption of the new bone tissue formed from periosteal cells in rabbits, but does not affect the bone tissue formed from human progenitor osteogenic periosteal cells. These functional characteristics of rabbit periosteal cells should be considered when planning the experiment.

Optical and Electron Microscopic Study of the Morphology and Ultrastructure of Biofilms Formed by Streptococcus pyogenes.

Our study confirmed the capacity of S. pyogenes strains to form biofilms on abiotic surfaces. Chains of streptococci surrounded by bluish film were seen under a microscope after alcian blue staining of the preparations grown on slides. On ultrathin sections in transmission electron microscope, the extracellular matrix (indicator of biofilm maturity) became visible after staining with alcian blue. Microscopy of the sections shows structures characteristic of a biofilm in spaces between the cells. Scanning electron microscopy also demonstrates the presence of a biomembrane. Importantly that type 1M strain forming in fact no membranes when cultured on plastic plates (Costar) formed biofilms on the glass. It seems that the conditions for the biofilm formation on the plastic and on the glass differ, due to which the exopolymeric matrices formed on different surfaces vary by biochemical composition.

Effects of Miramistin and Phosprenil on Microbial Biofilms.

Effects of Miramistin and Phosprenil on biofilms of S. pyogenes, S. aureus, E. coli, L. acidophilus, and L. plantarum were studied. Significant differences in the effects of these substances on mature biofilms of microorganisms and the process of their formation were observed. Miramistin had significant inhibiting effects on the forming of biofilms and on the formed biofilms of all studied microorganisms. Treatment with Miramistin inhibited biofilm formation by 2-3 times compared to the control. This effect was found already after using of Miramistin in the low doses (3.12 µg/ml). Inhibition of the growth of a formed biofilm was observed only after treatment with Miramistin in the high doses (25-50 µg/ml). Phosprenil in the high doses (15-30 mg/ml) inhibited the forming of biofilms, especially the biofilms of S. pyogenes and L. plantarum (by 3-4.5 times). Treatment of formed biofilms with the agent in doses of 6.0 and 0.6 mg/ml was associated with pronounced stimulation of its growth in S. pyogenes, S. aureus, and L. acidophilus.

[The application of the computer technologies for the mathematical simulation of the ethmoidal labyrinth]. / Komp'iuternye tekhnologii dlia matematicheskogo modelirovaniia reshetchatogo labirinta.

The objective of the present work was to study the relationship between the dimensions of the ethmoidal labyrinth and the skull in the subjects differing in the nose shape by means of the factorial and correlation analysis with the application of the modern computer-assisted methods for the three-dimensional reconstruction of the skull. We developed an original method for computed craniometry with the use the original program that made it possible to determine the standard intravital craniometrics characteristics of the human skull with a high degree of accuracy based on the results of analysis of 200 computed tomograms of the head. It was shown that the length of the inferior turbinated bones and the posterior edge of the orbital plate is of special relevance for practically all parameters of the ethmoidal labyrinth. Also, the width of the choanae positively relates to the height of the ethmoidal labyrinth.

Interleukin-6 in Sera of Mice Inoculated with Group A Streptococcus and Cultural Supernatants.

Dynamics of IL-6 level was studied in blood serum of CBA mice receiving intraperitoneal injections of killed and live culture of group A Streptococcus and its supernatants. It was found that administration of killed culture was followed by a significant rise in IL-6 level (by 4.7 times in 1 h and by 9.3 times in 5 h in comparison with the control). By 24 h, cytokine content was below the control. The highest levels of IL-6 were found after treatment with supernatants of Streptococcus cultures (by 10.5 times in 1 h and by 14.9 times in 5 h, in comparison with the control). Administration of live culture was accompanied by an increase in IL-6 concentration by 3.2 times in 3 h. In this experimental series, the maximum level of IL-6 was found in 48 h (by 5.2 times), and then it gradually decreased below the control. Different dynamics of changes in IL-6 level after administration of killed and live cultures of group A Streptococcus may suggests that they activate different signal pathways.

[Molecular genetic mechanisms of drug resistance in prostate cancer].

The major problem in prostate cancer treatment is the development of drug resistance and especially important, cross-resistance. The mechanisms of drug resistance, which are divided into ligand-dependent (requiring the presence of androgens in the cell) and independent (not requiring the presence of androgens) are reviewed. The mechanisms are mainly represented with mutations of the androgen receptor and expression of aberrant constitutively active splice variants, as well as up-regulation of genes involved in androgens synthesis.

[Downregulation of OGDHL expression is associated with promoter hypermethylation in colorectal cancer].

Cell metabolic reprogramming is one of the cancer hallmarks. Glycolysis activation, along with suppression of oxidative phosphorylation and, to a lower extent, the TCA cycle, occurs in the majority of malignant tumors. A bioinformatics search for the glucose metabolism genes that are differentially expressed in colorectal cancer (CC) was performed using the data of The Cancer Genome Atlas (TCGA) Project. OGDHL for an oxoglutarate dehydrogenase complex subunit, which is involved in the TCA cycle and is indirectly responsible for the induction of apoptosis, was identified as one of the most promising candidates. A quantitative PCR analysis showed, on average, an eightfold downregulation of OGDHL in 50% (15/30) of CC samples. Based on the TCGA data, promoter hypermethylation was assumed to be a major mechanism of OGDHL inactivation. Bisulfite sequencing identified the OGDHL promoter region (+327 ... +767 relative to the transcription start site) that is often methylated in CC samples with downregulated ODGHL expression (80%, 8/10) and is possibly crucial for gene inactivation. Thus, frequent and significant OGDHL downregulation due to hypermethylation of a specific promoter region was demonstrated for CC. The OGDHL promoter methylation pattern was assumed to provide a marker for differential diagnosis of CIMP+ (CpG island methylator phenotype) tumors, which display dense hypermethylation of the promoter region in many genes.

Successive Administration of Streptococcus Type 5 Group A Antigens and S. typhimurium Antigenic Complex Corrects Elevation of Serum Cytokine Concentration and Number of Bone Marrow Stromal Pluripotent Cells in CBA Mice Induced by Each Antigen Separately.

Administration of bacterial antigens to CBA mice induced an increase in serum concentration of virtually all cytokines with a peak in 4 h after administration of S. typhimurium antigens and in 7 h after administration of streptococcus antigens. In 20 h, cytokine concentrations returned to the control level or were slightly below it. In 4 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, we observed a significant decrease in serum concentrations of IFN-γ, IL-10, GM-CSF, IL-12, and TNF-α, in comparison with injection S. typhimurium antigens alone and IL-5, IL-10, GM-CSF, and TNF-α in comparison with injection of streptococcus antigens alone; the concentrations of IL-2 and IFN-γ, in contrast, increased by 1.5 times in this case. In 20 h after administration of S. typhimurium antigens, the number of multipotential stromal cells (MSC) in the bone marrow and their cloning efficiency (ECF-MSC) increased by 4.8 and 4.4 times, respectively, in comparison with the control, while after administration of streptococcus antigens by 2.6 and 2.4 times, respectively. In 20 h after administration of S. typhimurium antigens preceded 3 h before by administration of streptococcus antigens, these parameters increased by 3.2 and 2.9 times, respectively, in comparison with the control, i.e. the observed increase in the level of MSC count and ECF-MSC is more consistent with the response of the stromal tissue to streptococcus antigens. Thus, successive administration of two bacterial antigens corrected both serum cytokine profiles and MSC response to administration of each antigen separately, which indicates changeability of the stromal tissue in response to changes in the immune response.

Changed Serum Cytokine Profile in Mice in Response to Streptococcus A Culture.

Comparative analysis of serum cytokine profiles of CBA mice was carried out 1, 5, 24, and 48 h after intraperitoneal injection of killed culture of different streptococcus A types. The production of cytokines in response to different streptococcus types varied. The highest level was recorded in response to types 1M and 3T+M, more often detected in invasive streptococcal infection. The highest levels of IL-2 were recorded in response to 1M (47-fold increase in comparison with the control) and 3T+M streptococcus types (more than 10-fold increase). Injections of these types also led to an increase of IFN-γ level (15.6 and 11.3 times, respectively). The level of TNF-α increased less (3.6 times in response to 3T+M and 2.6 times in response to 1M type). The levels of IL-5, IL-10, and IL-12 increased 2-3-fold. Injections of 1T and 5M types led to just a 2-fold increase of cytokine levels. These data indicated induction of the immune response trend by mainly Th1 or mixed Th1/Th2 pattern in response to group A streptococcus antigens.

Effects of combined treatment with complex S. typhimurium antigens and factors stimulating osteogenesis (curettage, BMP-2) on multipotent bone marrow stromal cells and serum concentration of cytokines in CBA mice.

The content of multipotent stromal cells (MSC) in the bone marrow and efficiency of their cloning (ECF-MSC) increased by 3 times 1 day after administration of complex S. typhimurium antigens to CBA mice, while the relative content of alkaline phosphatase-positive MSC colonies (marker of osteogenesis; P(+) colonies) decreased from 14% (control) to 3%. After administration of the complex S. typhimurium antigens to CBA mice 3 h after (or 3 h before) curettage or treatment with morphogenetic protein (BMP-2), the content of MSC and ECF-MSC decreased on the next day by ~3 times in comparison with animals receiving antigens alone and approached the control level. The relative content of P(+) colonies increased to 20 and 35%, respectively, in comparison with animals receiving antigens (3%), but was significantly lower than after curettage (34%) or BMP-2 (42%) administration. Expression of IL-1ß, IL-6, IL-12, TNF-α, and IFN-γ genes in the primary cultures of stromal bone marrow cells induced by antigen administration was suppressed, while the concentrations of IL-12 and TNF-α in the culture medium sharply decreased after antigen treatment in combination with curettage or BMP-2 administration. Administration of complex S. typhimurium antigens after pretreatment with BMP-2 (3 h before) was associated with a decrease in serum levels of IL-2, IFN-γ, IL-12, and TNF-α in mice receiving BMP-2+S. typhimurium group 4 h after treatment in comparison with the animals receiving only S. typhimurium antigens alone by 1.9, 4.4, 1.5, and 6 times, respectively, i.e. to normal level or below it, while the concentration of IL-10 increased by almost 2 times, which probably reflected anti-inflammatory properties of BMP-2. These data probably attest to competitive relations between osteogenesis and immune response at the level of MSC.

[Tumor stem cells in breast cancer].

High mortality from breast cancer is associated with the high heterogeneity of tumor and the frequent recurrences of the pathological process, which are due to the presence of tumor stem cells. The review considers the biological properties of tumor stem cells, the molecular mechanisms of their regulation, interaction with the microenvironment, and their role in the heterogeneity of the morphological and clinical forms of breast cancer.

[Biofilm formation by group A Streptococci of various types and study of antibiotics effect on this process].

AIM: Evaluation of the ability to form biofilms by various M, T and MT-types of group A streptococci (GAS), as well as study of the effect of various antibiotics on biofilm formation. MATERIALS AND METHOD: 43 strains of various M and T type GAS were studied. The cultures were grown in Todd-Hewitt broth with the addition of 0.5% yeast extract. Comparative evaluation of the ability to form biofilm was carried out using photometry. Benzylpenicillin, oxacillin, cepha- losporin, cefuroxime and ceftriaxone antibiotics were used at various concentrations. RESULTS: GAS differ significantly by their ability to form biofilms. The highest ability was noted in 8 strains--2M, 9M, 12M, 13M, 19M, 30M, 36M-types and 6MT type. Simultaneous introduction of GAS cultures and antibiotics into the culture well, except for ceftriaxone, resulted in growth inhibition of both plankton cells and biofilms. CONCLUSION: The ability of GAS to form biofilm depends on streptococci serotype. During simultaneous introduction of GAS with antibiotics into the well, the biofilm does not form.

[Features of aphasic disorders in patients with acute cerebrovascular disorder in the posterior cerebral artery basin]. / Osobennosti afaticheskikh rasstroistv u patsientov s ostrym narusheniem mozgovogo krovoobrashcheniya v basseine zadnei mozgovoi arterii.

Aphasia is a systemic disorder of formed speech that develops as a result of local brain lesions. Most aphasias are characterized by damage to secondary cortical fields, which in turn are responsible for the performance of the functions of gnosis and praxis, which explains the variability in the manifestations of speech disorders in patients with acute cerebrovascular accidents. However, it is necessary in each case to diagnose the central pathological mechanism, which underlies the development of the entire syndrome and determines the entire clinical picture. The most important task of a speech therapist-aphasiologist is to qualify the defect, namely to isolate the mechanism and analyze the syndrome in order to select individual methods of corrective restoration. This article presents a case of a patient with an ischemic stroke in the left posterior cerebral artery with the development of amnestic aphasia in combination with alexia without agraphia.

Effect of BMP-2 protein on the count and osteogenic properties of multipotent stromal cells and expression of cytokine genes in primary cultures of bone marrow and spleen cells from CBA mice immunized with bacterial antigens.

We studied the effect of BMP-2 added to the culture medium on osteogenic and proliferative properties of multipotent stromal cells (MSC) and on the expression of cytokine genes induced by immunization of experimental animals with bacterial antigens. It is shown that the presence of BMP-2 in the culture medium stimulates proliferation of bone marrow MSC and especially spleen MSC (which was seen from enlargement of MSC colonies); improves the efficiency of MSC cloning; increases osteogenic activity of mouse bone marrow MSC; induces osteogenic differentiation of splenic MSC (osteogenesis is normally not observed in the spleen); reduces the number of macrophages in cultures; inhibits synthesis of mRNA for proinflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α) that typically occurs in cultures of the bone marrow and spleen from animals immunized with S. typhimurium or group A streptococcus antigens. Bearing in mind that proinflammatory cytokines negatively affect osteogenic activity of the bone marrow, we can hypothesize that BMP-2 not only stimulates osteogenesis, but also provides optimal conditions for its realization by suppressing the expression of genes encoding these cytokines.