[Personalized cell therapy for early postoperative bullous keratopathy (experimental proof and clinical results)].

The article presents the results of a long-term research on development and clinical application of personalized cell therapy (PCT) for treatment of early postoperative (manifesting within the first 3 months after surgery) bullous keratopathy (BK). The method of intracameral PCT implies in vitro incubation of the patient's blood sample with poly(A:U) stimulator, separation of the serum with activated leukocytes, and injection of the final cell preparation into the anterior chamber. The fundamental part of the research was aimed at a detailed description of the cell preparation and investigation of its possible mechanisms of action. Cytokine and growth factor level in the cell preparation suggested that its high clinical efficacy might be due to its ability to improve regeneration of damaged corneal endothelium. The clinical study was conducted on a group of 52 patients with early BK. A significant effect (smoothing of the Descement's membrane folds, complete resorption of corneal edema, improvement of corneal transparency, reduction of corneal thickness and increase of visual acuity by 0.49 +/- 0.27) was achieved in 44.2% of patients, while partial effect was seen in 21.1% of patients. There was no clinical effect in 34.6% of patients. In those patients who developed significant or partial clinical effect after the PCT, many endotheliocytes appeared to have multiple nuclei (2 and more). In some patients polyploid nuclei persisted for 3-5 years after the treatment. Polyploidy results from incomplete mitosis which might be due to regenerative processes in the endothelium stimulated by the PCT. Obviously, high efficacy and relative simplicity of the method should promote its further clinical introduction.

[Study of the three-dimensional structure and biointegrative characteristics of porous orbital implant materials].

Plastic repair operations in ophthalmic surgery frequently require the use of various biological and artificial implants. The biointegrative properties of implants are provided by the physicochemical characteristics of the material that is used to make them, by the three-dimensional structure of an implant and its immunological characteristics. The authors comparatively studied the three-dimensional structure of a Carbotexim-M carbonic felt, porous polyethylene, polytetrafluoroethylene, demineralized bone alloimplant, and marine coral hydroxyapatite, which are employed in orbital surgery and also examined the adhesion of fibroblasts to their surface. Comparative study using scanning electron microscopy could reveal the specific features of the relief and parameters of the threshold space of the study implant materials. A method was developed to estimate cell adhesion to the implant materials, by taking into account three-dimensional structure of their surface.

[Local express auto-cytokine therapy (a complex of cytokines) in the treatment of viral and nonviral eye lesions]. / Lokal'naia ékspress-autotsitokinoterapiia (kompleks tsitokinov) v lechenii virusnykh i nevirusnykh porazhenii glaz.

The method of local express-autocytocinetherapy (LEACCT) consists in using an experimentally tested autologous complex of cytokines (alpha- beta- gamma-interferons, interleukins 2.8, tumor necrosis factor gamma etc.), which is produced by joining the autoblood of patients with immune-modulator poludan (complex poly A:poly U). The external LEACCT and the intrachamber LEACCT are distinguished. The former presupposes the administration (subconjunctivally and in installations) of the autoblood-poludan mixture. This method is used in herpes- and adenovirus keratoconjunctivitis, slow re-epithelization after laser keratectomy and in eye burns (178 patients). The latter comprises, apart from the external LEACCT procedures, a 1-4-time introduction of the mentioned mixture into patient's anterior chamber. It was used in endothelial herpetic keratoiridocyclitis, initial bullous keratopathy, severe keratoconus and in injuries of the anterior lens capsule (117 patients). The clinical-study results (main group--295 patients) show that the designed method based on poludan has pronounced anti-inflammatory and regenerative effects in different viral and virus-free lesions of the eye tissues; it is simple and can be applied in clinical settings. An increased visual acuity ranging from 0.05 to 1.0 was registered in 85% of cases. The action of the complex of cytokines is as close as possible to the physiological mechanism, which provides for avoiding the side-effects and for applying the treatment scheme in situations when the administration of other drugs is limited or their efficiency is not sufficient.

[Theoretical validation of local express auto-cytokine therapy]. / K voprosu o teoreticheskom obosnovanii metodiki lokal'noi ékspress-autotsitokinoterapii.

Theoretical validation for local express autocytokine therapy (LEACKT), used in recent years with good results for the treatment of viral and nonviral diseases of the eyes, is provided. In vitro experiments showed that autologous cytokine complex appearing after stimulation of patient's peripheral whole blood with poludan (polyA:polyY complex, 200 micrograms) includes all three interferon types (alpha, beta, and gamma) and interleukin-8 (IL-8) and tumor necrosis factor (TNF). It was shown for the first time that poludan stimulated the production of IL-8, the main chemotactic cytokine inducing chemotaxis, neutrophil activation, angiogenesis stimulation, and regeneration. This was not paralleled by an increase in the production of TNF, the key mediator of inflammation, whose level in whole blood did not increase. Addition of poludan to cell culture led to a 2-fold decrease of lymphocyte apoptosis in comparison with that after treatment with dexamethasone alone. The results of experiments indicate that LEACKT with poludan is characterized by pronounced antiinflammatory, interferonogenic, regeneratory, and antiapoptosis effects in various diseases of the anterior segment of the eye and is a simple and safe method.

[Role and significance of structural, metabolic and functional disorders of mononuclear phagocytes in pathogenesis of caseous pneumonia]. / Rol' i znachenie strukturno-metabolicheskikh i funktsional'nykh naryshenii fagotsitarnoi sistemy v patogeneze kazeoznoi pnevmonii.

Comprehensive clinical, X-ray, cytochemical, morphological, biochemical, and immunological studies of 14 patients with caseous pneumonia have provided evidence that significant structural, metabolic, and functional disorders of mononuclear phagocytes (MNP) play a leading role in the pathogenesis of acute tuberculosis. Structural and metabolic disorders of macrophages and monocytes in patients with caseous pneumonia result from impaired mitochondrial oxidation and glycolysis, aggregation and latinization of the membranes of lysosomes, release of their contents into the cytosol with damages to intracellular structures and the cellular membrane itself. This is also suggested by a drastic rise in the production of prostaglandins E2 and F2 alpha, prostaglandins E2 in particular, in the supernatants of cultured monocytes (100 nM). This is determined as the membrane-damaging effect of MNP due to the toxic action of rapidly multiplying mycobacterial population not only in the lung, but even in blood. MNP structural and metabolic disturbances are an equivalent to their lowered functional activity, as evidenced by a considerable deficiency of synthesis of intracellular and secretory pools of interleukin I and by a fall in their migrational and adhesive activities, two thirds of macrophages having signs of dystrophy and cytolysis. On entering the specific inflammatory area of the lung, these cells abundantly disintegrate. Their destruction leads to the elaboration of enzymes, prostaglandins, and other biologically active agents, which promotes the occurrence of extensive caseously destructive changes and creates conditions for rapid multiplication of mycobacteria.

Contact Effects of Mononuclear Cells on L929 Fibroblast Morphological Phenotype.

We studied the contact interactions of human peripheral blood mononuclear cells (MNC) and transformed mouse fibroblast cell line L929 (L-cells), namely their effects on morphological phenotype of L-cells. The morphological characteristics of the fibroblast, (cell area, nucleus-cytoplasmic ratio, cell spreading, cell shape) were estimated with the aid of fight scanning microscopy, followed by computer image analysis. Contact interaction between fibroblasts and MNC caused normalization of morphological phenotype of the fibroblasts (increase of cell area, shape factor, spreading and decrease of nucleus-cytoplasmic ratio). This phenomenon was revealed by analysis of both average morphological characteristics and population contents. Only a particular subpopulation of L-cells, but not the whole population, was shown to be normalized by the effects of MNC. For elucidation of responsible MNC population, which was capable of influencing on fibroblast morphological phenotype, we separated MNC in several cell types: adherent cells, non-adherent cells, which were separated in E-rosetting cells, and non-E-rosetting cells. Only non-adherent and E-rosetting cells could normalize the morphological phenotype. E-rosetting population consisted of 85% of CD3(+) (T lymphocytes) and 15percnt; of CD56(+)/CD16(+) (natural killers). Supernatants of MNC, and MNC cocultured L-cells, obtained after 24 h incubation in the cell culture medium, were not able to normalize the morphological phenotype of the fibroblasts. They had small denormalizing effect on the fibroblasts (decrease of cell area, shape factor, spreading and increase of nucleus-cytoplasmic ratio). The results of this study indicate that the contact interaction between MNC and fibroblasts may normalize transformed fibroblast morphological phenotype. The dependence of fibroblast functional state on their morphological phenotype imply the presence of regulatory mechanism of contact interaction between fibroblasts and MNC, which determines many live processes in fibroblast.

[Phenotype of peripheral blood lymphocytes of healthy donors and patients with corneal allotransplantation under conditions of hyperbaric oxygenation]. / Fenotip limfotsitov perifericheskoi krovi zdorovykh donorov i bol'nykh s rogovichnym allotransplantatom v usloviiakh giperbaricheskoi oksigenatsii.

The purpose of this study was to elucidate the possible immunomodulating effect of hyperbaric oxygenation (HBO) on the immune status of patients with various ophthalmopathies and developing the graft-versus-host reaction after perforating keratoplasty. Surface phenotype of the peripheral blood lymphocytes was studied by two-color flow cytofluorometry in donors and patients with the rejection syndrome after transplantation of the cornea before and after a course of oxygen therapy. HBO exerted no immunodepressive effect, as judged from changes in the subpopulation composition (CD3+, CD4+, CD8+, CD16+, and CD56+) and expression of activation markers (CD25 and HLA-DR), in both patients and donors. The most probable explanation is that HBO effect on the immune system is indirect. On the other hand, from a clinical viewpoint the effect of HBO is undoubtedly positive, and hence, HBO exposure may be recommended for clinical application in transplantations of the cornea.

[The activation processes of T-lymphocytes and their regulatory subpopulations in patients with pulmonary tuberculosis]. / Sostoianie protsessov aktivatsii T-limfotsitov i ikh reguliatornykh subpopuliatsii u bol'nykh tuberkulezom legkikh.

Immunophenotyping of lymphocytes by direct double-color immunofluorescence, investigation of metabolic activity of succinate dehydrogenase, alpha-glycerophosphate dehydrogenase, glucose-6-phosphate dehydrogenase, NAD- and NADP-diaphorases were performed in 77 tuberculous patients and 50 healthy donors. Activation of T-lymphocytes and their regulatory subpopulations is shown to depend on the extension of lung infiltration. At the same time, there were marked disorders in intracellular metabolism of peripheral blood lymphocytes. Close correlation exists between the activity of lymphocytic energy enzymes and the degree of expression on lymphocytes of receptors to interleukin-2, transferrin and HLA-DR-molecules. Cytochemical reactions for evaluation of blood lymphocyte energetic enzymes activity may be used in clinical practice for characterization of activation of T-lymphocytes and their regulatory subpopulations.

[Increased apoptosis of immunocompetent cells as a possible mechanism in the development of immunodeficiency in patients with acutely progressive tuberculosis]. / Povyshennyi apoptoz immunokompetentnykh kletok kak odin iz vozmozhnykh mekhanizmov v razvitii immunodefitsita u bol'nykh ostroptogressiruiushchim tuberkulezom.

The formation of immunodeficiency whose likely mechanism is apoptosis of some immunocompetent cells was studied in 35 patients with caseous pneumonia. The leading clinical sign of apoptosis in acutely progressive tuberculosis is significant lymphopenia (4-10%). Immunological studies indicated a substantial reduction in the count of T lymphocytes and their regulatory subpopulations of different phenotypes. In vitro mitogenic induction causes a decline of activated CD3+, CD4+, and CD8+ cells on the average by 10-40%, a decrease in the proliferative and synthetic functions, as compared with those in patients with infiltrative tuberculosis of the lung and healthy donors. Cytochemical findings in the same patients show that half the lymphocytes in the blood samples from patients with caseous pneumonia has profound intracellular metabolic disturbances. These cells are unavailable and undergo apoptosis, which determines immunodeficiency in patients with acutely progressive tuberculosis.

[A comparative study of the expression of activation markers on T-lymphocyte subpopulations in different forms of bronchial asthma]. / Sravnitel'noe izuchenie ékspressii markerov aktivatsii na subpopuliatsiiakh T-limfotsitov pri razlichnykh formakh bronkhial'noi astmy.

In this study we investigated a phenotype of intact and PHA-stimulated T-lymphocytes from peripheral blood of patients with allergic and nonallergic asthma. Expression of the activation markers (CD25, CD71, HLA-DR) on subsets of T-lymphocytes (CD4+ and CD8+) was determined using double-colour flow cytometry. It was found that nonallergic patients had increased percentages of CD4+CD25+, CD8+CD25+, CD4+HLA-DR+, CD8+HLA-DR+ cells. Allergic patients had a significant increase of CD4+CD25+ cells only. To examine functional significance of these changes mononuclear cells were cultivated with PHA in vitro. Thus, our findings improve our knowledge about asthma pathogenesis and create the grounds for pathogenetically validated immunocorrection.

[Cellular and intracellular markers of lymphocyte activation in patients with infiltrative pulmonary tuberculosis]. / Kletochnye i vnutrikletochnye markery aktivatsii limfotsitov u bol'nykh infil'trativnym tuberkulezom legkikh.

The authors investigated markers of activation of CD25-, CD71- and HLD-DR molecules on CD3+, CD4+ and CD8+ lymphocytes simultaneously with cytochemical activity of the main energetic and synthetic enzymes in 34 patients with new-onset infiltrative tuberculosis of the lungs and in 30 healthy controls. It was found that reduced number of effector and regulatory lymphocyte subpopulations was recorded only in patients with advanced infiltrative tuberculosis in line with a sharp decline in enzymatic and functional activity of immunocompetent cells. In local infiltrative tuberculosis impairment of activation processes occurs only at intracellular level in time of energy metabolism shift to predominant glycolysis, this allowing lymphocytes to persist functionally active. A close relationship is shown between glycolytic enzymes and activation of CD3+ cells and their subpopulations. Basing on the above findings, one may refer the above enzymes to intracellular activation markers of lymphocytes.

[Complex assessment of the production of 3 variants of interleukin-1 by human peripheral blood monocytes]. / Kompleksnaia otsenka produktsii 3 variantov interleikina-1 monotsitami perifericheskoi krovi cheloveka.

In vitro production of intracellular, membrane-associated and secreted interleukin-1 was investigated by peripheral blood monocytes from healthy donors. Activity of three IL-1 variants was assayed by the proliferation of responsive C3H/HeJ mouse thymocytes. Complex evaluation of IL-1 pools production by human PBM would provide a key to a better understanding of various diseases pathogenesis.

[Assessment of the activity of tumor necrosis factor in normal state and in ovarian tumors]. / Otsenka aktivnosti faktora nekroza opukholi v norme i pri opukholiakh iaichnikov.

The cytotoxic activity of tumor necrosis factor of healthy individuals and patients with cystadenoma and adenocarcinoma of ovaries was investigated. Tumor necrosis factor (TNF) spontaneous activities of healthy donors and patients with cystadenoma of ovaries was the same. The level of TNF spontaneous activity of patients with adenocarcinoma was increased. LPS-induced TNF cytotoxicity in patients with cystadenoma was decreased. Mononuclear cells of patients with adenocarcinoma did not respond to LPS stimulation by increased TNF production.

[Assessment of the functional activity of lymphokine-activated killer cells in healthy donors and in patients with interleukin-2-dependent immunodeficiency]. / Otsenka funktsional'noi aktivnosti limfokinaktivirovannykh killerov u zdorovykh donorov i bol'nykh s interleikin-2-zavisimym immunodefitsitom.

The methodological aspects of Lak-cells functional activity test were determined. NK-resistance of P-815 culture cells and NK-sensitive K-562 cells were used as target cells. The highest cytotoxicity level was registered at the 5-th day of culturing MNC with rIL-2 at the final concentration (100 U/ml). The method developed was used for testing healthy donors group and group of patients with IL-2 dependent immunodeficiency (ophthalmic herpes). Data obtained show the significant decrease of LAK-cells functional activity of patients in comparison with healthy donors. Thus, the developed method is recommended for complex estimation of immune status of patients with IL-2-dependent immuno deficiency.

[Functional activity of natural killers and lymphocyte antibody-dependent cytotoxicity in patients with acute disorders of the cerebral circulation]. / Funktsional'naia aktivnost' estestvennykh killerov i antitelozavisimaia tsitoksichnost' limfotsitov u bol'nykh s ostrymi narusheniiami mozgovogo krovoobrashcheniia.

Dynamic examination of 47 patients over the first three weeks after the development of ischemic stroke (IS) has revealed a significant decrease in the natural killer activity (NKA) and antibody-dependent cellular cytotoxicity (ADCC). A decrease in the NK activity in IS patients appears to be related to dyslipoproteinemia, which has been confirmed by the detection of a reverse correlation between the values of the NKA and the atherogenicity coefficient, while suppression of ADCC in the same patients is explained by the suppressive influence of the prostaglandin system, which has been investigated in vitro. The elucidation in IS patients of immunopathological phenomena related to impairments of the effector component of immunity and of activities of NK- and K-cells justifies the inclusion of immunocorrecting drugs into the comprehensive therapy of IS.