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.

[Epithelial-stromal interactions in pancreatic adenocarcinoma: the role of stroma in disease progression]. / Epitelial'no-stromal'nye vzaimodeistviya v adenokartsinome podzheludochnoi zhelezy: rol' stromy v progressirovanii zabolevaniya.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and difficult to treat form of pancreas cancer. PDAC and other solid cancers contain both tumor cells and normal connective tissue cells called stromal cells, which are responsible for the excess production of extracellular matrix. It is known that in more than 90% of PDAC tumors and in many other types of cancer, mutations of the KRAS gene are observed, the reciprocal signaling of which has been shown between tumor and stromal cells in vitro. Pancreatic stromal stellate cells are considered precursors of activated or tumor-associated fibroblasts (CAFs), which are an increasing population of cells that proliferate in situ or are recruited into the tumor. CAFs are a heterogeneous population of stromal fibroblasts with different molecular profiles that change during tumorigenesis. Both immunosuppressive and immunosuppressive subsets of CAFs can coexist in the stroma of a single tumor. Based on the heterogeneity of the intertumor stroma, attempts are being made to classify PDAC and predict the course of the disease.

[Is there a claudin-low phenotype of breast cancer?] / Sushchestvuet li «nizkoklaudinovyi¼ fenotip raka molochnoi zhelezy?

The novel molecular subtype of breast cancer (BC), named "claudin-low", was described in 2007. It was characterized by the consistently low expression of genes involved in the formation of epithelial tight junctions in combination with the high activation of genes associated with the epithelial-to-mesenchymal transition, as well as tumor stem cell markers. The similar claudin- low subtype was later identified at the transcriptional level in bladder cancer, gastric cancer, and serous ovarian cancer. However, only in relation to BC, attempts were made to create a surrogate panel for immunohistochemical identification of this subtype in a manner like the intrinsic molecular BC subtypes identified using three main markers, such as ER, PR, and HER-2. At the same time, the ambiguity in the expression of claudins among the subtypes of BC, which is defined by various authors at the immunohistochemical level, as well as the absence of both the confirmed set of immunohistochemical criteria and a unified approach to their assessment, complicate these efforts. The purpose of the review is to show that the immunohistochemical identification of claudin-low subtype of BC is a separate problem that has significant limitations, needs standardization and has not yet reached diagnostic value.

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.

[Whooping cough in different periods of pregnancy].

AIM: To study the clinical course, diagnosis of pertussis in pregnant women. MATERIALS AND METHODS: Under observation were 25 pregnant women with whooping cough. The age structure was dominated by patients from 6 to 35 years old, amounting to 64%. At the time of hospitalization in 2 (8%) patients the gestation period corresponded to the first trimester, in 7 (28%) II, in 9 (36%) III. In 7 (28%) patients, the diagnosis of whooping cough was made during childbirth. The following methods were used to verify pertussis: bacteriological, PCR and serological (ELISA). RESULTS AND DISCUSSION: Clinical analysis showed that pregnant women with pertussis who came to the hospital were admitted to hospital mainly in the late stages of the disease (at 45 week in 68.6% of cases). Pertussis infection in all cases was typical: in moderate form 96%, mild at 4%. In 64% of patients, whooping cough accompanied by other respiratory diseases, which were the main cause of development of bronchitis. The use of ELISA allowed to confirm the clinical diagnosis in 91.7% of patients. CONCLUSION: The course of whooping cough in pregnant women is characterized by a typical symptom complex. To improve the verification of the diagnosis, it is advisable to use modern laboratory tests (PCR, ELISA).

A Simplified Streptozotocin-Induced Diabetes Model in Nude Mice.

Preclinical studies of human cellular and tissue-based products (HCT/Ps) for transplantation therapy of type 1 diabetes mellitus (T1DM) necessarily involve animal models, particularly mouse models of diabetes induced by streptozotocin (STZ). These models should mimic the clinical and metabolic manifestations of T1DM in humans (face validity) and be similar to T1DM in terms of the pathogenetic mechanism (construct validity). Furthermore, since HCT/Ps contain human cells, modeling of diabetes in immune-deficient animals is obligatory. Here we describe the most simplified diabetes model in Nude mice. Diabetes was induced in 31 males by a single intraperitoneal injection of STZ in normal saline at a medium-to-high dose of 150 mg/kg body weight. Fourteen control animals received only saline. Non-fasting plasma glucose (PG) levels were measured periodically for 50 days. All STZ-treated mice survived beyond 50 days. By day 15 after STZ administration, 22 of 31 (71%) mice developed stable diabetes based on the following criteria: (1) non-fasting PG ≥ 15 mmol/L on consecutive measurements up until day 50; (2) no diabetes remission. The mean non-fasting PG in mice with stable diabetes over the period of 35 days was equal to 25.7 mmol/L. On day 50, mean plasma insulin concentration, mean pancreatic insulin content, and the average number of ß-cells in pancreatic islets were 2.6, 8.4, and 50 times lower, respectively, than in the control animals. We consider that our Nude mouse model of diabetes meets face validity and construct validity criteria and can be used in preclinical studies of HCT/Ps.

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.

[Role of tenascin С in triple-negative breast cancer]. / Rol' tenastsina S pri troinom negativnom rake molochnoi zhelezy.

AIM: to establish a relationship between the main markers tumor stem cells (TSCs), CD44, and CD24, the level of tenascin C production, and chemoresistance in triple-negative breast cancer (BC). SUBJECTS AND METHODS: Thirty biopsy specimens from triple-negative BC patients who had conventionally received preoperative chemotherapy followed by surgery were selected in the investigation. All the selected patients were conventionally assigned to neoadjuvant polychemotherapy (PCT) with paclitaxel and carboplatin. The surgical specimens were analyzed in relation to the degree of a tumor morphological response to PCT. The magnitude of the health-promoting effect of neoadjuvant therapy was evaluated according to the residual cancer burden (RCB) system using an on-line calculator; RCB was categorized into classes (from RCB-0 to RCB-III). The markers CD44, СD24, and tenascin C were identified by the standard immunoperoxidase method in the primary biopsies. RESULTS: Varying morphological responses of triple-negative breast cancer to PCT were revealed, which showed resistance in 60% of the cases. The chemoresistance found in most (87%) cases coincided with the identification of the CD44+/CD24low/- profile. The detection of the higher production of the extracellular matrix tenascin C participating in the formation of the TSC niche fully combined with the CD44+/CD24low/- phenotype; while the maximum response to tenascin C was noted in the cases differing in not only a lack of responses to PCTs, but also in the most aggressive course in conjunction with metastatic disease. CONCLUSION: Immunohistochemical analysis shows that the unique association between the CD44+/CD24low/- phenotype and the pronounced production of tenascin C may have a prognostic potential, prospectively indicating the inefficiency of neoadjuvant PCT, in particular that with platinum derivatives, which is used for the standard treatment of triple-negative BC. Taking into account the role of tenascin C in invasion, metastasis, and chemoresistance, it per se may be considered as a promising target for the targeted and/or combined therapy of triple-negative BC.

[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.

[Clinical features of pertussis in adults].

AIM: To study the clinical features of pertussis in adults. SUBJECTS AND METHODS: The clinical manifestations of pertussis in adults were analyzed in 62 patients treated in the Core Unit and Obstetric Unit, Clinical Infectious Diseases Hospital One, Moscow Healthcare Department, in 2011-2013. RESULTS: Investigations conducted in the familial foci of pertussis showed that its incidence rate was 23.7 and 10% in adults and in pediatric institutions, respectively. The source of infant infection was adults in almost one third of cases, mothers in 72% and fathers in 27.7%. Assessment of the symptom complex of pertussis in the sick mothers in the observed period ascertained that 94±3% had experienced the typical form of whooping cough: 72.0±5.7 and 22.0±5.3% had its mild and moderate forms, respectively. The disease had run a latent course in 6±3% of the patients. The paper describes cases of pertussis in 5 pregnant women admitted to the obstetric unit for complaints of persistent cough. The diagnosis of whooping cough in all the cases was based on clinical and epidemiological findings and serological tests showing specific IgA and IgG antibodies. Three and two patients experienced moderate and mild pertussis, respectively. CONCLUSION: Pertussis now remains one of the infectious diseases relevant not only to a pediatric population, but also to an adult one. Adult pertussis maintains the main clinical features typical for this infection with a predominance of its mild forms, which makes its diagnosis difficult. A serological test (enzyme immunoassay) is of high diagnostic value for verifying whooping cough in adults under the present conditions.

[Isolation of circulating tumor cells in blood by means of "Isolation by SizE of Tumor cells (ISET)"].

There is evidence of the importance of circulating tumor cells in bloodstream as a factor of poor prognosis of cancer. The optimum method for isolating and studying of these cells is not defined. The most common methods are either based on the isolation of tumor genetic material from blood or on immune-mediated isolation of epithelial tumor cells. The first group of methods is characterized by a lack of specificity, while the latter do not allow identifying a pool of cells undergone in bloodstream epithelial-mesenchymal transformation. There is presented an overview of results of clinical trials of a new technique of isolation of tumor cells from bloodstream based on the patients' blood filtration through a membrane with defined pore sizes (ISET-Isolation by SizE of Tumor cells).

[Light optical and ultrastructural characteristics of human focal cortical dysplasias].

Focal cortical dysplasias (FCD) are one of the most common causes of drug-resistant epilepsy. Morphological changes in the focal cortical dysplasia tissues obtained from epileptic patients during surgical interventions were analyzed. Eleven specimens of the cerebral cortex with FCD areas were explored. There were a variety of light optical and ultrastructural abnormalities affecting different portions of the cortical arrangement, as well as a considerable heterogeneity in these changes. Special emphasis was placed on morphological correlations between excitation and inhibition processes when possible mechanisms responsible for the epileptogenicity of dysplasia cells were assessed. The findings suggest that there is a preponderance of FCD tissue excitation processes along with depressed inhibition processes.

[Isotypes of anti-pertussis antibodies in pertussis patients at different stages of the disease].

AIM: Evaluation of anti-pertussis antibodies in pertussis patients at different stages after the onset of the disease. MATERIALS AND METHODS: Levels of IgG, IgG1, IgG2, IgG3, IgG4, IgA and IgM antibodies against the antigen complex of pertussis were evaluated by enzyme immunoassay (EIA). Sera samples were analyzed from 208 pertussis patients examined from week 1 to 10 after the onset of the disease. RESULTS: 51%, 82% and 86% pertussis patients, and 67%, 72% and 78% patients examined from week 1 to 3 after the onset of the disease had increased levels of IgM, IgA and IgG antibodies respectively. 85%, 70%, 74% and 68% pertussis patients, and 76%, 57%, 87%, 57% patients examined from week 1 to 3 after the onset of the disease had increased IgG1, IgG2, IgG3 and IgG4 levels respectively. 92% of all examined pertussis patients and 83% of patients examined from week 1 to 3 after the onset of the disease had an overall increase of anti-pertussis antibody levels. Increased level of IgM antibodies was detected predominately from week 1 to 5 after the onset of the disease. Most of the patients examined from week 3 to 10 after the onset of the disease had increased levels of IgA, IgG, IgG1, IgG2 and IgG4 antibodies, and IgG3 antibody level was increased predominately in patients examined from week 2 to 6 after the onset of the disease. CONCLUSION: Serological indicators of pertussis measured by EIA were observed in 83% of the patients examined at the early stages after the onset of the disease. Simultaneous measurement of IgA, IgG and IgM antibody levels is the most effective approach for serological diagnostics of pertussis due anti-pertussis antibodies isotype composition heterogeneity at different stages after the onset of the disease. Increased levels of IgM and IgG3 antibodies are serologic indicators of the acute phase of pertussis infection.

[A direct accelerated method for detection of a whooping cough pathogen].

The developed direct method for the laboratory diagnosis of pertussis, which is based on isothermal amplification technologies, has a high (100%) specificity and sensitivity (102 m.cl.), can detect the pathogen of the disease just in the clinical sample from a patient within 7-8 hours after start of the study. The clinical trials conducted at Infectious Diseases Hospital One (Moscow) on examination of 103 patients (63 patients with the clinical diagnosis of pertussis and 40 with other respiratory tract diseases) provided evidence its high specificity and diagnostic efficiency as compared with a bacteriological test, the efficiency in different clinical types of the disease and during examinations of patients in different periods after the onset of the disease, as well as during examinations of patients with suspected pertussis and pertussis-like diseases.

[Molecular factors of angiogenesis in renal tissue of patients with chronic glomerulonephritis: association with nephrosclerosis and anemia].

AIM: To study correlations between accumulation of angiogenesis molecular factors (hypoxia-inducible factor-1alpha - HIF-1alpha, vascular endothelial growth factor - VEGF, thrombospondin - TSP-1) in kidney biopsy tissue from chronic glomerulonephritis (CGN) patients and severity of nephrosclerosis, obliteration of renal capillary bed, filtration dysfunction and anemia. MATERIAL AND METHODS: We examined 22 patients with marked proteinuria (2.77; 5.7, mean 4.2 g/ day). Half of the patients had nephrotic syndrome. Glomerular filtration rate (GFR) by Cochroft-Golt formula was 68 (53;84) ml/min/1.73 m2. According to renal biopsy findings, CGN was detected in 19 patients, 2 patients had lupus nephritis (LN), 1 patient had renal amyloidosis. Nineteen CGN patients were divided into two groups by nephrosclerosis severity: group 1-7 patients with moderate nephrosclerosis, group 2-12 patients with severe nephrosclerosis. Cryostate sections of renal biopsy tissue samples were studied immunohistochemically using monoclonal antibodies to HIF-1alpha, VEGF, TSP-1, CD34. The reaction intensity was assessed by 6-point scale semiquantitative method. RESULTS: Response to HIF-1alpha was stronger in the tubular epithelium than in glomeruli. No correlation was observed between accumulation of HIF-1alpha in the glomeruli and tubular epithelium. Intensity of glomerular staining correlated with severity of proteinuria (Rs = 0.63, p < 0.05), intensity of HIF-1alpha accumulation in tubular epithelium correlated with duration of the kidney disease (Rs = 0.74, p < 0.001), duration of persistent arterial hypertension (Rs = 0.68, p < 0.05) and severity of nephrosclerosis. VEGF and TSP-1 were found in equal quantity both in the glomeruli and renal interstitium. CGN patients with marked nephrosclerosis had lower accumulation of VEGF and higher TSP-1 in the interstitium. No correlation was found between intensity of tubular epithelium response to HIF-1alpha and accumulation of VEGF in the interstitium. Patients with severe nephrosclerosis demonstrated weaker staining of tubulointerstitium to CD34, reflecting the degree of its vascularisation. Significant correlation between CD34 and expression of HIF-1alpha, VEGF, TSP-1 was not registered. In patients with low intensity of tubular epithelial staining to HIF-1alpha (less than 2 points) anemia was detected in 63% versus 18% in patients with more intensive accumulation. CONCLUSION: CGN progression is associated with development of renal tubulointerstitial ischemia. High tubular production of HIF-1alpha was not accompanied with activation of VEGF accumulation in renal interstitium but was associated with reduced risk of anemia in CGN patients with manifest nephrosclerosis.

[Antigenic composition and serologic characteristics of domestic acellular pertussis vaccine].

AIM: To assess antigenic composition consistency and serological characteristics of domestic acellular pertussis vaccine. MATERIALS AND METHODS: Amount of pertussis toxin, filamentous hemagglutinin, agglutinogens types 1, 2, and 3 in experimental batches of vaccine was measured by enzyme immunoassay. Levels of antibodies to aforementioned antigens as well as to lipopolysaccbaride in serum samples obtained from patients with pertussis and healthy vaccinated children were measured by the same method. The amount of lypopolysaccharide was determined by LAL test. RESULTS: Studied batches of vaccine were standard on amount of all protein antigens as well as lipopolysaccharide. Spectrum of antibodies to vaccine components in serum samples from patients with pertussis and healthy vaccinated children included antibodies to individual antigens: pertussis toxin, filamentous hemagglutinin, lipopolysaccharide, agglutinogens types 1, 2, and 3. CONCLUSION: Developed technology for manufacturing acellular pertussis vaccine allows to consistently produce preparations with standard amount of all components. Vaccine components interact with antibodies to wide spectrum of B. pertussis antigens.

[Correlation of p16/INK4a gene damages and protein expression in the tumor tissue of sporadic breast cancer].

A tumor emerges due to the structural and functional abnormalities occurring in the genes, which causes a change in the spectrum of protein molecules. Strong correlations between the gene damages and following changes in the protein spectrum make it possible to study different stages of carcinogenesis and to create a more complete system of molecular markers for the diagnosis of different types of tumors, which comprises protein markers and DNA markers. The present investigation has studied a correlation between the inactivation mechanisms (structural and functional) of the suppressor gene p16/INK4a, which occur at the level of DNA, and the results of its protein expression examined by immunohistochemical methods in the tumor specimens from patients with breast cancer. The investigation could indicate that p16/INK4a gene damage frequently occurred in the tumors of the above type. In the majority of study cases, molecular damages revealed in the gene diminish on its protein expression; however, there are still cases that defy generally accepted explanations.

[Frequent allelic losses in tumor-associated stromal cells and tumor epitelium of prostate cancer].

It has become increasingly clear that tumor microenvironment plays a critical role in carcinogenesis. Accumulation of genetic alterations is typical not only for cancer epithelial cells but tumor-associated fibroblasts as well. Tumor epithelia, tumor-associated stroma from prostatectomy specimens of patients with prostate cancer and cells from prostatic intraepithelial neoplasia (PIN) and adjacent stroma from males with PIN were isolated by using laser capture microdissection. Microsatellite allelotyping was evaluated using 4 highly polymorphic markers for chromosomal regions 8p22, 16q23-24 and 13q14. Incidences of alterations (loss of heterozygosity or allelic imbalance) were 48% for region 8p22, 72% for 16q23 and 37% for 13q14. The LOH frequencies in tumor-associated stroma cells were very similar. Alterations at chromosome 13q were significantly associated with advanced tumor stage, whereas AI at 16q was also associated with high Gleason score and lymph node metastasis. We find some incidences of allelic imbalance in premalignant lesions in epithelial (16-27%) and stromal (7-22%) components. Our results show that the frequencies of genetic aberrations are as high in stromal cells as in tumor cells.

[The specific features of the expression of E-cadherin and beta-catenin in adenomyosis].

The expression of E-cadherin and beta-catenin in the eutopic and ectopic endometrium was studied in 40 patients with adenomyosis and 12 without this condition, by using an immunohistochemical test and enzyme immunoassay. There was increased expression of E-cadherin and beta-catenin in the eutopic and ectopic endometrium in adenomyosis. The cytoplasmic expression of beta-catenin was also revealed in the smooth muscle cells surrounding the foci of adenomyosis. The E-cadherin concentration measured by enzyme immunoassay was significantly higher in the endometrium and myometrium of patients with adenomyosis than in the controls. There was a higher expression of this protein with a longer duration of the disease. Thus, the formation of ectopic foci in adenomyosis may be argued to be unassociated with the decreased adhesion of epithelial cells. On the contrary, the authors documented the enhanced adhesion of epithelial cells in the ectopic foci, which was likely to be adaptive due to the altered microcirculation.

[Transforming growth factor as a marker beta of nephrogenetic disturbance in congenital obstructive uropathies].

The paper presents the results of morphological and immunohistochemical studies of the kidneys and ureters obtained at surgery in 25 children aged from 2 days to 3 years who had congenital obstructive uropathies. The morphological study revealed varying renal tissue dysplasia concurrent with dysplasia of the prepelvic or distal ureter. Immunohistochemically, the kidneys showed a pronounced expression of TGFbeta1, TGF(1R1 and R2 in the nephrocytes of non-differentiated and collecting tubules, in the tubular lumen, vascular walls, in the interstitial tissue and cell interstitial infiltrates, on the hypoplastic and disoriented ureteral smooth muscle fibers. The use of the ILIAS-method to determine urinary TGF levels before and after surgery indicated a direct correlation of these indices with the degree of renal tissue dysplasia.