[Analysis of genetic determinants of multidrug and extensively drug-resistant Mycobacterium tuberculosis using oligonucleotide microchip].

Steadily growing resistance of the tuberculosis causative agent towards a broad spectrum of anti-tuberculosis drugs calls for rapid and reliable methods for identifying the genetic determinants responsible for this resistance. In this study, we present a biochip-based method for simultaneous identification of mutations within rpoB gene associated with rifampin resistance, mutations in katG, inhA, ahpC genes responsible for isoniazid resistance, mutations within the regions of gyrA and gyrB genes leading to fluoroquinolones resistance, and mutations in the rrs gene and the eis promoter region associated with the resistance to kanamycin, capreomycin and amikacin. The oligonucleotide microchip, as the core element of this assay, provides simultaneous identification of 99 mutations in the format "one sample--one PCR--one microchip", and it makes it possible to complete analysis of multi-drug-resistant and extensively drug-resistant tuberculosis within a single day. The tests on 63 Mycobacterium tuberculosis clinical isolates with different resistance profiles using the developed approach allows us to reveal the spectrum of drug-resistance associated mutations, and to estimate the significance of the inclusion of extra genetic loci in the determination of M. tuberculosis drug resistance.

[Molecular and epidemiology studies of HIV-1 prevalence in the Republic of Sakha (Yakutia)].

The gag, pol, and env genomic regions of HIV-1 variants currently circulating in the Republic of Sakha (Yakutia) were analyzed. The results of the study indicated that in this area there were HIV-1 variants belonging to two subtypes: A (IDU-A) and B, the former being predominant in this area and in the Russian Federation. The IDU-B-East strain was first isolated from a heterosexually infected patient, which suggests that the strain is outside the risk group of injection drug users. No cases of primary infection with resistant variants were notified during the study.

Nanostructure of vortex during explosion welding.

The microstructure of a bimetallic joint made by explosion welding of orthorhombic titanium aluminide (Ti-30Al-16Nb-1Zr-1Mo) with commercially pure titanium is studied. It is found that the welded joint has a multilayered structure including a severely deformed zone observed in both materials, a recrystallized zone of titanium, and a transition zone near the interface. Typical elements of the transition zone-a wavy interface, macrorotations of the lattice, vortices and tracks of fragments of the initial materials-are determined. It is shown that the observed vortices are formed most probably due to local melting of the material near the contact surface. Evidence for this assumption is deduced from the presence of dipoles, which consist of two vortices of different helicity and an ultrafine duplex structure of the vortex. Also, high mixing of the material near the vortex is only possible by the turbulent transport whose coefficient is several orders of magnitude larger than the coefficient of atomic diffusion in liquids. The role played by fragmentation in both the formation of lattice macrorotations and the passage of coarse particles of one material through the bulk of the other is determined.

Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

Detection of mutations in Mycobacterium tuberculosis genome determining resistance to fluoroquinolones by hybridization on biological microchips.

We developed a method of identification of Mycobacterium tuberculosis with simultaneous evaluation of the sensitivity to fluoroquinolones on a biological microchip array. The method of multiplex two-staged PCR followed by hybridization of a biochip makes it possible to detect 8 mutant variants of gyrA gene occurring in fluoroquinolone-resistant strains (approximately 85% all resistant forms) within 1 day. Using this method we analyzed 107 cultures isolated from patients with tuberculosis and 78 sputum samples. Mutations in gyrA gene were detected in 48 (92%) resistant strains. Natural S95T polymorphism in gyrA gene was detected in all resistant and in 76% sensitive strains. The sensitivity and specificity of the proposed method calculated on the basis of the analysis of sputum samples (n=78) were 94 and 100%, respectively.

[Use of molecular-biological microchip TB-BIOCHIP-2 for detecting of Mycobacterium tuberculosis with multidrug resistance to fluoroquinolones in patients with new detected and chronic tuberculosis].

At present the left-handed "respiratory" quinolones such as moxifloxacin and levofloxacin are the most promising drugs for therapy of multidrug resistant tuberculosis (MDR). Fast and specific diagnostics of sensitivity of M. tuberculosis (MBT) with MDR to this group of drugs is required for timely prescription of adequate chemotherapy and its correction in case of MBT resistance to fluoroquinolones. A new generation of biological microchips - TB-BIOCHIP-2 makes possible to detect 9 mutation types in quinolones resistant determination region (QRDR) of gene. About 800 samples from 169 patients in Antituberculosis center were studied. In patients with new detected tuberculosis 23.5% MBT resistant to isoniazid and rifampicin (MDR) and sensitive to fluoroquinolones were revealed. In patients with chronic tuberculosis 65.5% MBT-MDR were revealed. Our results were confirmed with detecting ofloxacin resistance on Lowenstein - Jensen. In addition efficiency of TB-BIOCHIP-2 to control drug testing sensitivity of MBT-MDR on fluoroquinolones was confirmed.

[New indodicarbocyanine dyes for the biological microchip technology].

New indodicarbocyanine dyes with the carboxybutyl group in position 3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.

[Lipid peroxidation in the pathogenesis of primary open-angle glaucoma]. / Perekisnoe okislenie lipidov v patogeneze pervichnoi otkrytougol'noi glaukomy.

19 tissue samplings of the iris and trabecula from 17 patients, aged 49 to 79, with primary open-angle glaucoma (POAG) of different stages were examined for the purpose of studying the condition of lipid membranes in the iridial and trabecular tissues by electron microscopy. The results are indicative of that the key role, in the POAG pathogenesis, belongs to lipid peroxidation of posterior and anterior leaves of the retinal pigment epithelium, which is an activator of apoptotic processes in the trabecular endothelium. Therefore, it is advisable to use the lipid- and water-soluble antioxidative drugs to correct the physiological antioxidative deficiency within a complex POAG treatment.

[Local senile ocular amyloidosis in the pathogenesis of open-angle glaucoma and pseudo-exfoliative syndrome]. / Lokal'nyi starcheskii amiloidoz glaza v patogeneze otkrytougol'noi glaukomy i psevdoéksfoliativnogo sindroma.

A total of 173 biopsy samples of the iris and drainage apparatus obtained at anti-glaucoma operations from 115 patients, aged 32 to 90, with primary open-angle glaucoma and pseudoexfoliative syndrome of different severity degrees were morphologically examined. Similar anatomofunction embryological peculiarities of the eye and brain create prerequisites for the development, in the eye, of processes analogical to Alzheimer's disease. Amyloid angiopathy of the iris is a key element of ischemia, which triggers the mechanisms of apoptosis and lipid peroxidation in the pigment epithelium membranes; this process is accompanied by an intensified synthesis and conformation of fibrous structures of tauprotein and beta-amyloid. Defradation of melanin and proteoglycans leads to the formation of neurotoxic metabolites. The above processes result in suppressing the protective antioxidant reactions as well as in blocking, by fibrous structures, the cellular cytoplasm and extracellular matrix.

[The causes of necrobiosis and apoptosis of corneal epithelial cells during primary acquired keratoconus]. / Prichiny nekrobioza i apoptoza épitelial'nykh kletok rogovitsy pri pervichnom priobretennom keratokonuse rogovitsy.

We studied 56 biopsy samples of conjunctiva and 50 corneal discs excised from 28 patients with acquired keratoconus cornea. The conjunctivas in all biopsy samples showed various stages of immune inflammation. Necrobiotic changes have been revealed in epithelium of the corneal discs going by the pathways of apoptosis--programmed cell death--and oncosis--initial edematic stage of necrobiosis. At the stage of acute inflammation they are due to cytotoxic effect of the lymphocytes, monocytes, and macrophages. Antibody-dependent cytotoxicity mediated by plasma and lymphoid cells predominates at this stage. At the reparative stage of inflammation ischemia, an inductor of apoptosis and oncosis, underlies necrobiotic changes in corneal epithelium.

[Cell-to-cell interactions between iris and trabecular meshwork in primary open-angle glaucoma]. / Kletochnye vzaimodeistviia raduzhnoi obolochki i trabekuliarnogo apparata glaza pri otkrytougol'noi glaukome.

Pathomorphological study of the ocular draining system in the presence of an ischemic iris was carried out in patients with primary open-angle glaucoma (POAG). A total of 173 biopsy specimens of the sclera and iris were collected during anti-glaucoma operations in 115 patients aged 32-90 years. The specimens were stained for amyloid with Congo red, thioflavin, and toluidine blue at pH 4.6 and 5.8 for detecting glycosaminoglycanes. The specimens were examined by histochemical and electron microscopic methods. Morphological changes in the trabecular system in POAG are due to abnormalities in the iridal microcirculatory bed. The status of the trabecular system during the initial and advanced stages of glaucoma depends on the severity of involvement of iridal arterioles, capillaries, and venules, modulating the biochemical processes in the anterior segment of the eye. Endotheliocyte and collagen matrix pathology and subsequent degradation of the trabecular system are closely related to dystrophic processes in the iris (release of plasma protein from the vessels and melanin granules into anterior chamber humor, cytotoxic effects of degradation products on adjacent tissues). The detected changes imply the use of drugs improving blood supply to the eye at the microcirculatory level and antihypoxants and antioxidants in local and total-systems therapy of patients with POAG.

[The role of the mast cells of the conjunctiva in the intercellular interactions in keratoconus and epithelial-endothelial corneal dystrophy]. / Rol' tuchnykh kletok kon''iunktivy glaza v mezhkletochnykh vzaimodeistviiakh pri keratokonuse i épitelial'no-éndotelial'noi distrofii rogovitsy.

The aim of this study was assessment of the role of mast cells in inflammatory processes associated with keratoconus and corneal epithelial-endothelial dystrophy and clinical assessment of Lecrolin, a specially designed dosage form (eye drops) of potassium cromoglycate (intal) used as an antiallergic drug in asthma, rhinitis, etc. Examinations of 43 biopsy specimens of the conjunctiva from patients with keratoconus and epithelial-endothelial corneal dystrophy demonstrated the significance of immune inflammation in the pathogenesis of these diseases. Mast cells are a typical cell element participating in inflammation at all stages. The count of mast cells increases from 2.5 during the acute stage to 8.86 during the chronic stage, which is explained by absence of the leukocytic stage and proteolysis and stabilization of mature labrocyte degranulation associated with it. Reactions of mast cells with lymphocytes and monocytes/macrophages lead to proliferation of fibroblasts and chronic development of collagen formation. Close relationships between microvessels and mast cells promote stable spasms of the vessels, impairing the microcirculatory bed and leading to ischemia of the anterior segment of the eye. In such cases sodium cromolyne drugs (lecrolin) inhibiting histamine release and stabilizing mast cell membranes are recommended.

[Morphogenesis of corneal epithelial-endothelial dystrophy (a comparative morphological analysis of corneal disks and biopsy specimens of the conjunctiva)]. / Morfogenez épitelial'no-éndotelial'noi distrofii rogovitsy (sravnitel'no-morfologicheskii analiz rogovichnykh diskov i bioptatov kon"iunktivy).

The aim of this study was morphological analysis of corneal disks and conjunctival biopsy specimens in order to disclose the mechanisms of development of epithelial-endothelial dystrophy (EED) and validating pathogenetically-based therapy. Twenty-five patients were observed, 11 of these with implanted intraocular lenses of different localization. EED developed for 1 to 5 years. Sixteen biopsy specimens of the conjunctiva and 21 corneal disks were examined. The composition and index of inflammatory infiltrate, status and compactness of conjunctival vessels, severity of fibrosis, and mast cell degranulation were assessed. Mast cells play a positive role at the initial stages of tissue and cellular response to injury by regulating the formation of extracellular matrix and fibroblast proliferation. At the reparative stages of immune inflammation of the conjunctiva, mast cells are virtually the only cell population; fibrosis and sclerosis develop when the capillary bed is reduced, causing ischemic involvement of the anterior segment of the eye and dystrophic processes in the cornea. A differentiated approach to treatment of EED at the stages of immune inflammation and fibrosis is emphasized.

[The cellular aspects of amyloid angiopathies of the iris in open-angle glaucoma]. / Kletochnye aspekty amiloidnykh angiopatii raduzhnoi obolochki glaza pri otkrytougol'noi glaukome.

We studied cellular aspects of amyloid angiopathy of the iris upon decompensation of ocular pressure and the role of microcirculatory pathology in pathogenesis of the open angle glaucoma. A total of 115 iris biopsies were studied, which were taken during antiglaucoma operations and stained by Congo rot and thioflavine for amyloid and Toluidine blue for glycosaminoglycans. The biopsies from nine patients were studied at the ultrastructural level. Intravasal increase of the concentration of preamyloid proteins during spasms of the arterioles and capillaries should be considered as a provoking moment of the iris amyloidogenesis. Blockade of reticular structures of the microcirculatory basal membranes by preamyloid serum proteins, which provides for shedding of amyloid fibrils, dystrophic changes and detachment of the endothelial cells from the basal membrane, obliteration of lumens of the capillaries and arterioles, disturbed integrity of the microvascular barrier and accumulation of preamyloid and amyloid proteins in the extracellular matrix, ischemia and focal microinfarctions of the iris, and dystrophia of the melanocytes, are subsequent key factors. Amyloid angiopathy of the iris is a cause of ischemia of the anterior eye part, thus creating conditions for increased intraocular pressure and glaucoma.

[Morphological features of senile and secondary amyloidosis of the iris and sclera in patients with glaucoma]. / Morfologicheskie osobennosti starcheskogo i vtorichnogo amiloidoza raduzhki i sklery u bol'nykh glaukomoi.

Morphologic analysis of 173 biopsy specimens of the iris and sclera removed during antiglaucoma surgery in 115 patients with open-angle glaucoma revealed in 44.4% cases extracellular depositions of amyloid, which aggravated the course of glaucomatous process and were pathogenetically significant. Amyloidosis was revealed in the sclera in 82% cases and in the iris in 70%. Amyloid was detected by Congo red staining with control in polarized light and by toluidine blue staining for chondroitin and dermatan sulfates. Vascular and muscular (involving the pupil muscles) forms of amyloidosis were detected in the iris, pericollagen and focal forms in the draining system and sclera. The authors discuss the contribution of amyloidosis to the pathogenesis of glaucoma. These findings confirm M.M. Krasnov's concept about glaucoma polymorphism.

[Amyloidosis of the iris and sclera in the pathogenesis of glaucoma]. / Amiloidoz raduzhki i sklery v patogeneze glaukomy.

Morphological analysis of 173 biopsies of the iris and sclera removed during antiglaucoma operations in 115 patients with open angle glaucoma showed extracellular deposits of amyloid in 44.4% of cases, which complicated the course of glaucomatous process and had pathogenetic importance. Amyloid was identified by Congo red [correction of Congo-rot] staining with the control in the polarized light and also by toluidine blue staining for chondroitin and dermatan sulfates. The vascular and muscle (pupillary muscle) forms of amyloidosis were found in the iris and the pericollagen and focal forms in the drainage system and sclera. We propose to classify the amyloid form of glaucoma with specific morphological picture.

[The characteristics of the nucleolar organizer in tumors of the choroid of the eye]. / Kharakteristika iadryshkogo organizatora v opukholiakh sosudistoi obolochki glaza.

26 intraocular melanomas were studied histologically, ultrastructurally and morphometrically. For the first time the impregnation method was used for visualization of the nucleolar organizer (NO) in the cells of eye tumors using silver salts on the deparaffined sections. The efficiency and reliability of this method in the determination of the degree of malignancy of intraocular melanomas are demonstrated. A correlation existed between long-term postoperative results and the nucleolar organizer index (NO number/number of nuclei). The method can be practiced in any pathohistologic laboratory.

[Diagnostic biopsy of the conjunctiva in keratoconus]. / Diagnosticheskaia biopsiia kon''iunktivy pri keratokonuse.

Morphologic examinations of biopsy specimens of the conjunctiva from 28 patients with keratoconus (56 biopsy specimens) revealed two types of pathologic processes: (1) acute and chronic immune inflammations of cell-mediated and mixed type (in 9 patients) and (2) fibrosis and acellular sclerosis as the result of the inflammation with the reduction of the microcirculatory bed and ischemia (19 patients). These data indicate the necessity of a differentiated approach to pre- and postoperative drug therapy of patients with keratoconus, exposed to perforating keratoplasty. The clinical picture does not permit a differentiation between types and phases of the inflammation for objective individualized prescription of drug therapy before and after perforating keratoplasty, therefore conjunctival biopsy is advisable in such cases as a diagnostic test. Histologic analysis gives data on the migration and proliferation of inflammatory cells, on the production of the intercellular substance, on the microcirculatory status. The results of this study helped the authors suggest a schematic approach to conservative therapy of patients suffering from keratoconus, aimed at inhibition of the inflammation.

[Erythrocyte membranes during malignant growth]. / Membrany éritrotsitov pri zlokachestvennom roste.

Suspensions of red cells from normal subjects and cancer patients were incubated in the presence of hydrogen peroxide. The incubation gave rise to a flash of chemiluminescence including rapid and slow components. The concentration dependencies of chemiluminescence were plotted as was the dependence of chemiluminescence on the temperature of the suspension under test. The differences were found between chemiluminescence of donors' red cells and that of patients'. It is inferred that red cell membranes of cancer patients show both the decreased membrane resistance to the damage-inducing action of hydrogen peroxide and elevation of antioxidant activity.