[New criteria of effectiveness of anti-VEGF therapy in exudative age-related macular degeneration]. / Novye kriterii effektivnosti anti-VEGF-terapii pri ekssudativnoi forme vozrastnoi makulyarnoi degeneratsii.

OBJECTIVE: To evaluate the effectiveness of anti-angiogenic therapy using fluctuation and variability in patients with exudative age-related macular degeneration (AMD). MATERIAL AND METHODS: This study included 200 patients with types 1, 2, 3 neovascularization and polypoidal choroidal vasculopathy (PCV). All patients underwent standard ophthalmological examination, as well optical coherence tomography (OCT) and OCT angiography (OCT-A). Patients were divided into 4 quartiles based on fluctuation and variability. All patients underwent intravitreal administration of the anti-VEGF drug aflibercept (Eylea) manufactured by Bayer, Germany, using the Treat-and-Extend regimen. RESULTS: As a result of the study, a significant increase in visual acuity was revealed on the second year of treatment in patients in the 2nd and 3rd quartiles, best corrected visual acuity (BCVA) decreased in the 1st and 4th quartiles. In the group with paracentral fluctuation, a significant increase in BCVA by the end of the second year of treatment was observed in the 1st (p=0.05) and 3rd quartiles. As a result of a 2-year follow-up, it was found that BCVA values were lower in patients with the greatest variability (2nd, 3rd, and 4th quartiles). A significant increase in BCVA was observed in the 1st quartile (p=0.047). The largest number of patients with subretinal fibrosis that had fluctuations in the central zone and peripheral fluctuations was observed in the 4th quartile, the smallest - in the 2nd quartile. Similar pattern was observed in the variability group. In the setting of anti-VEGF therapy, a significant increase in BCVA was observed in patients with the lowest variability - in the 1st quartile (p=0.047), but in patients with fluctuations the highest BCVA values were observed in the 2nd and 3rd quartiles, while in the 4th quartile BCVA decreased (p=0.0562). CONCLUSIONS: The results of this study indicate that when treating patients with neovascular AMD, clinicians should aim for a treatment strategy that minimizes fluctuation and variability.

[Clinical case of MELAS syndrom]. / Klinicheskii sluchai sindroma MELAS.

Clinical case of mitochondrial encephalomyopathy manifested with lactic acidosis and stroke-like episodes was presented. The patient diagnosis was performed in childhood, based on clinical manifestation, and was confirmed with molecular genetic test (mutation m.3243A>G in gene MT-TL1 was revealed). Appropriate patient management required united efforts of different medical specialists with simultaneous administration of different drugs, modulating intracellular energy production. Due to contemporary medical science achievements, life expectancy of patients with mitochondrial diseases increases, and in age 18 such patients should be treated by adult-practice physicians. Due to such pathology rare incidence the adult-practice medical practitioners are insufficiently informed about principles of mitochondrial disease treatment, that has negative influence on patients condition.

[Effectiveness of anti-VEGF therapy for neovascular age-related macular degeneration in different types of exudative fluid localization]. / Effektivnost' anti-VEGF-terapii neovaskulyarnoi formy vozrastnoi makulyarnoi degeneratsii pri razlichnykh tipakh lokalizatsii ekssudativnoi zhidkosti.

PURPOSE: To assess the effectiveness of anti-VEGF therapy in different types of fluids localization in neovascular age-related macular degeneration (AMD). MATERIAL AND METHODS: The study included 16 people (16 eyes) with exudative AMD. The study included patients with fibrovascular detachment of the retinal pigment epithelium (RPE), subretinal and intraretinal fluid. The patients were divided into two groups: in the first group, fibrovascular RPE detachment was combined with subretinal fluid (9 eyes); in the second group, fibrovascular RPE detachment was accompanied by both sub- and intraretinal fluid (7 eyes). All patients underwent standard ophthalmological examination, which included visometry, biomicroscopy, and ophthalmoscopy under conditions of drug-induced mydriasis. Additionally, OCT of the macular region and OCT-angiography were performed. RESULTS: Over the entire observation period, the first group of patients received an average of 5.11 intravitreal injections (IVI), the second group - 5.14 IVI. Visual acuity was comparable in both groups at the beginning of the study. Subsequently, the treatment resulted in an increase in visual acuity after 3 months in the first (p=0.066) and second (p=0.043) groups, as well as after 12 months in both groups (p=0.043). In the first group of patients, after 12 months, an increase in RPE detachment was observed (p=0.942), which suggests that the disease activity remains underestimated on OCT when RPE detachment and subretinal fluid are combined. In the first group, complete resorption of subretinal fluid occurred in 2 people. In the second group, resorption of subretinal and intraretinal fluid occurred in 6 people. CONCLUSION: Regardless of the subtype of fluid, anti-VEGF therapy is an effective method for treating exudative AMD. Intravitreal injections are necessary both in the presence of intra- and/or subretinal fluid, and fluid under RPE. The greatest difficulty is assessment of the fluid under the RPE.

[Antiangiogenic therapy for types I and II macular neovascularization in age-related macular degeneration]. / Antiangiogennaya terapiya pri I i II podtipakh makulyarnoi neovaskulyarizatsii pri vozrastnoi makulyarnoi degeneratsii.

The neovascular form of age-related macular degeneration (AMD) is characterized by growth of newly formed vessels, accumulation of fluid and, in most cases, presence of retinal pigment epithelium detachment. Depending on its localization in relation to retinal pigment epithelium (RPE), macular neovascularization (MNV) can be considered type 1 when it is located under the RPE, and type 2 when it is invading the RPE and the neurosensory part of the retina. PURPOSE: To conduct a retrospective analysis of the use of anti-VEGF therapy in AMD patients with types I and II of MNV. MATERIAL AND METHODS: The study enrolled 89 AMD patients (89 eyes) with active MNV who have been under observation for 3 years. In the course of treatment all patients underwent standard ophthalmological examination that included visometry, biomicroscopy and ophthalmoscopy with mydriasis, as well as optical coherence tomography. RESULTS: Anti-VEGF therapy was found to stabilize best corrected visual acuity (BCVA) in both types of MNV (I and II). Comparison of the intraretinal and subretinal fluids (IRF and SRF) revealed that initially neuroepithelium detachment is more frequent (approximately in 90% of eyes) than IRF (30-40%). Antiangiogenic therapy is associated with better resorption of SRF, by the third year of the follow-up the neuroepithelium detachment is visualized in 60% of patients, while IRF remains and is observed in 40% of cases. CONCLUSION: Antiangiogenic therapy has shown good functional and morphological effectiveness in both first and second types of MNV.

[Noncoding Polymorphism rs6832151 Is an Attractive Candidate for Genome Editing Aimed at Finding New Molecular Mechanisms of Autoimmune Diseases].

Currently only a small fraction of the proteins encoded in the human genome serve as pharmaceutical targets. Genome-wide association studies are a powerful tool to uncover new genetic loci responsible for predisposition to complex diseases, such as autoimmune disorders. However, further work is still required to identify causative single-nucleotide polymorphisms (SNPs) which directly mediate the disease risk at these loci, and to determine their target genes. These genes can be located millions base pairs away from the regulatory SNPs. Here, by using bioinformatic tools and databases, we identified five intergenic autoimmunity-associated polymorphisms with high probability of being causative, for which the target genes are still unknown. We tested their ability to influence gene expression using luciferase reporter system. The polymorphism rs6832151 affected the reporter expression in the CEM human T-cell line upon the highest enhancer activity. Target genes of this SNP could be further identified by introducing point mutations to the genome and comparison of transcriptomes of the derivative cell sublines carrying alternative alleles of rs6832151.

[Real clinical practice of antiangiogenic therapy in patients with age-related macular degeneration. Retrospective analysis of functional results]. / Real'naya klinicheskaya praktika primeneniya antiangiogennoi terapii u patsientov s vozrastnoi makulyarnoi degeneratsiei. Retrospektivnyi analiz funktsional'nykh rezul'tatov.

PURPOSE: To assess the functional results of antiangiogenic therapy in patients with exudative form of age-related macular degeneration (AMD) in real clinical practice. MATERIAL AND METHODS: The study included 90 people (90 eyes) with active choroidal neovascularization (CNV) on the background of AMD. All patients were divided into 6 groups depending on the year of treatment - from 2013 to 2018, all patients were divided into 6 groups and overall the retrospective study sited at Research Institute of Eye Diseases (Moscow) lasted 8 years. All patients underwent standard ophthalmological examination including visometry, biomicroscopy and ophthalmoscopy under drug-induced mydriasis, as well as optical coherence tomography, fundus angiography and OCT-angiography. RESULTS: According to the results of the analysis of OCT data obtained from 2013 to 2017, among all patients with exudative AMD, patients with types I and II of CNV and single patients with RAP prevailed, which explains the high visual acuity - about 0.5 - in all groups after the start of the treatment (table 1 and 2). In 2018, 33.3% of patients were diagnosed with RAP (the same number of eyes as with types I and II of CNV), which can be explained by the introduction of OCT-angiography into wide clinical practice. The lack of increase in visual acuity is most likely associated with a small amount of intravitreal injections (IVI) - 4.8 IVI in the first year and 3.3 IVI in the second injection year. In patients who received more than three IVI in the first year of observation, visual acuity increased from 0.49±0.03 to 0.6±0.03 (p=0.04), in the case of less than three IVI in the first year, visual acuity was not changed, amounting to 0.42±0.1 before and 0.44±0.1 (p=0.655) after the treatment. CONCLUSIONS: Patients of all groups exhibited proportional stabilization of visual acuity, a decrease in the thickness of the retina and total macular volume. The lack of improvements of visual acuity is most likely associated with a small amount of IVI.

[Current understanding of pachychoroid spectrum diseases]. / Sovremennye predstavleniia o pakhikhorioidal'nykh zabolevaniiakh.

Pachychoroid spectrum diseases (the prefix 'pachy-' means 'thick') were proposed as the term indicating an abnormal increase in choroidal thickness, dilatation of choroid vessels and other structural changes in choroid architecture. Pachychoroid spectrum diseases include central serous chorioretinopathy, polypoidal choroidal vasculopathy, pachychoroid pigment epitheliopathy, focal choroidal excavation, pachychoroid neovasculopathy, peripapillary pachychoroid syndrome. Studying the new group of diseases associated with pachychoroid phenotype is a topical task in modern ophthalmology. The emergence of new diagnostic methods such as OCT with angiography mode allows detailed study of this disease group, helps determine the prognosis and choose the tactic for management of these patients.

Patient-centred care in an occupational medicine clinic.

BACKGROUND: Patient-centred care (PCC) has been associated with improved patient satisfaction outcomes in a variety of clinical settings. There is a paucity of research addressing the concept of PCC in an occupational medicine context. AIMS: To assess patient perception and compare physician and patient perceptions of patient centredness of the care at a specialty occupational medicine clinic. METHODS: An observational study design using the Patient Perception of Patient Centeredness Questionnaire (PPPC) at an ambulatory tertiary care occupational health clinic. Results were analysed using a standardized coding system. Summary scores were compared to results reported in a primary care setting. Patient and physician scores were compared to detect physician-patient differences in perceived patient centredness of care. RESULTS: Of 47 eligible patients 37 consented to participate and seven were excluded due to incomplete data. Summary scores of patient perceptions of patient centredness were similar but somewhat better than scores reported in a primary care setting. Perceived patient centredness of care was high and there was minimal discordance between patient and physician scores. CONCLUSIONS: This study demonstrated that PCC can be measured in an occupational health setting. In an ambulatory tertiary care occupational health clinic there was a high degree of patient centredness of care which may be explained by a variety of factors. Future research should consider whether similar findings exist in other occupational medicine practice settings.

Constitutive Expression of NRAS with Q61R Driver Mutation Activates Processes of Epithelial-Mesenchymal Transition and Leads to Substantial Transcriptome Change of Nthy-ori 3-1 Thyroid Epithelial Cells.

The Q61R mutation of the NRAS gene is one of the most frequent driver mutations of thyroid cancer. Tumors with this mutation are characterized by invasion into blood vessels and formation of distant metastases. To study the role of this mutation in the growth of thyroid cancer, we developed a model system on the basis of thyroid epithelial cell line Nthy-ori 3-1 transduced by a lentiviral vector containing the NRAS gene with the Q61R mutation. It was found that the expression of NRAS(Q61R) in thyroid epithelial cells has a profound influence on groups of genes involved in the formation of intercellular contacts, as well as in processes of epithelial-mesenchymal transition and cell invasion. The alteration in the expression of these genes affects the phenotype of the model cells, which acquire traits of mesenchymal cells and demonstrate increased ability for survival and growth without attachment to the substrate. The key regulators of these processes are transcription factors belonging to families SNAIL, ZEB, and TWIST, and in different types of tumors the contribution of each individual factor can vary greatly. In our model system, phenotype change correlates with an increase in the expression of SNAIL2 and TWIST2 factors, which indicates their possible role in regulating invasive growth of thyroid cancer with the mutation of NRAS(Q61R).

Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.

α-Synuclein knockout mice have cognitive impairments.

α-Synuclein is a member of the synuclein family of cytoplasmic, predominantly neuron-specific proteins. Considerable amount of α-synuclein is found in axons and presynaptic terminals of neurons located in brain areas responsible for emotions and memory. In the present study we have carried out behavioral evaluation of spatial and working long-term memory of α-synuclein knockout mice. Our data shows that α-synuclein knockout mice have reduced learning ability in tests requiring both working and spatial memory. For the first time we have demonstrated that α-synuclein is necessary for these types of learning.

Nonlinear isochrones in murine left ventricular pressure-volume loops: how well does the time-varying elastance concept hold?

The linear time-varying elastance theory is frequently used to describe the change in ventricular stiffness during the cardiac cycle. The concept assumes that all isochrones (i.e., curves that connect pressure-volume data occurring at the same time) are linear and have a common volume intercept. Of specific interest is the steepest isochrone, the end-systolic pressure-volume relationship (ESPVR), of which the slope serves as an index for cardiac contractile function. Pressure-volume measurements, achieved with a combined pressure-conductance catheter in the left ventricle of 13 open-chest anesthetized mice, showed a marked curvilinearity of the isochrones. We therefore analyzed the shape of the isochrones by using six regression algorithms (two linear, two quadratic, and two logarithmic, each with a fixed or time-varying intercept) and discussed the consequences for the elastance concept. Our main observations were 1) the volume intercept varies considerably with time; 2) isochrones are equally well described by using quadratic or logarithmic regression; 3) linear regression with a fixed intercept shows poor correlation (R(2) < 0.75) during isovolumic relaxation and early filling; and 4) logarithmic regression is superior in estimating the fixed volume intercept of the ESPVR. In conclusion, the linear time-varying elastance fails to provide a sufficiently robust model to account for changes in pressure and volume during the cardiac cycle in the mouse ventricle. A new framework accounting for the nonlinear shape of the isochrones needs to be developed.

Interleukin-12 receptor/STAT4 signaling is required for the development of autoimmune myocarditis in mice by an interferon-gamma-independent pathway.

BACKGROUND: Interleukin (IL)-12 exerts a potent proinflammatory effect by stimulating T-helper (Th) 1 responses. This effect is believed to be mediated primarily through the activation of STAT4 and subsequent production of interferon (IFN)-gamma. Methods and Results- We examined the role of IL-12 receptor (IL-12R) signaling in the development of murine experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization. Both IL-12Rbeta1-deficient mice and STAT4-deficient mice were resistant to the induction of myocarditis. Treatment with exogenous IL-12 exacerbated disease. We questioned whether IFN-gamma is required for the disease-promoting activity of IL-12. On the contrary, we found that IFN-gamma suppresses EAM. Lack of IFN-gamma due to either depletion with an antibody or a genetic deficiency exacerbated myocarditis. Spleens from IFN-gamma-deficient mice immunized with cardiac myosin showed increased cellularity; greater numbers of CD3+, CD4+, CD8+, and IL-2-producing cells; and heightened ability to produce cytokines on stimulation in vitro. Treatment of mice with recombinant IFN-gamma suppressed the development of myocarditis. CONCLUSIONS: IL-12/IL-12R/STAT4 signaling promotes the development of EAM. In contrast, IFN-gamma plays a protective role. The disease-limiting effects of IFN-gamma might be explained by its ability to control the expansion of activated T lymphocytes.

Contribution of the innate immune system to autoimmune myocarditis: a role for complement.

Myocarditis is a principal cause of heart disease among young adults and is often a precursor of heart failure due to dilated cardiomyopathy. We show here that complement is critical for the induction of experimental autoimmune myocarditis and that it acts through complement receptor type 1 (CR1) and type 2 (CR2). We also found a subset of CD44(hi)CD62L(lo) T cells that expresses CR1 and CR2 and propose that both receptors are involved in the expression of B and T cell activation markers, T cell proliferation and cytokine production. These findings provide a mechanism by which activated complement, a key product of the innate immune response, modulates the induction of an autoimmune disease.

Experimental autoimmune myocarditis in A/J mice is an interleukin-4-dependent disease with a Th2 phenotype.

Myocarditis in humans is often associated with an autoimmune process in which cardiac myosin (CM) is a major autoantigen. Experimental autoimmune myocarditis (EAM) is induced in mice by immunization with CM. We found that EAM in A/J mice exhibits a Th2-like phenotype demonstrated by the histological picture of the heart lesions (eosinophils and giant cells) and by the humoral response (association of IgG1 response with disease and up-regulation of total IgE). Blocking interleukin (IL)-4 with anti-IL-4 monoclonal antibody (mAb) reduced the severity of EAM. This reduction in severity was associated with a shift from a Th2-like to a Th1-like phenotype represented by a reduction in CM-specific IgG1; an increase in CM-specific IgG2a; an abrogation of total IgE response; a decrease in IL-4, IL-5, and IL-13; as well as a dramatic increase in interferon (IFN)-gamma production in vitro. Based on the latter finding, we hypothesized that IFN-gamma limits disease. Indeed, IFN-gamma blockade with a mAb exacerbated disease. The ameliorating effect of IL-4 blockade was abrogated by co-administration of anti-IFN-gamma mAb. Thus, EAM represents a model of an organ-specific autoimmune disease associated with a Th2 phenotype, in which IL-4 promotes the disease and IFN-gamma limits it. Suppression of IFN-gamma represents at least one of the mechanisms by which IL-4 promotes EAM.

Nasal administration of cardiac myosin suppresses autoimmune myocarditis in mice.

OBJECTIVES: This study was designed to examine whether myocarditis induced in a mouse model can be effectively suppressed by nasal administration of cardiac myosin (CM). BACKGROUND: Myocarditis in humans often follows viral infection and is accompanied by evidence of an autoimmune response to CM. Treatment has been hampered by the fact that measures undertaken to reduce the autoimmune response often enhance the viral infection. Delivery of antigen via nasal route has been shown to induce antigen-specific tolerance and suppress certain autoimmune diseases in animal models. METHODS: Myocarditis was induced in A/J mice by two subcutaneous injections of CM emulsified in complete Freund's adjuvant. Nasal instillation of CM (200 microg/mouse) or vehicle buffer was carried out three days before the first subcutaneous injection (day -3). The effect of nasal instillation of CM on cardiac histopathology, cytokine production by splenocytes, and antibody response was examined three weeks after the first subcutaneous injection (day 21). RESULTS: Nasal administration of CM effectively reduced the severity of myocarditis. Consistent with the histological findings, the levels of interleukin-2 (IL-2), tumor necrosis factor-alpha, and IL-1beta produced by splenocytes in response to CM were significantly decreased. In addition, the serum levels of IgE and IgG1 anti-myosin antibodies were suppressed. However, the levels of transforming growth factor-beta (TGF-beta) and CM-specific IgA antibodies were not affected. CONCLUSIONS: Taken together, our results do not support active suppression through upregulation of TGF-beta, IL-4, and IL-10 as a mechanism of tolerance, but favor anergy or deletion of both Th1 and Th2 autoreactive T cells.

Characterization of murine autoimmune myocarditis induced by self and foreign cardiac myosin.

Previously we showed that autoimmune myocarditis could be induced in mice by immunization with purified murine cardiac myosin (MCM). In this study, we found that identical disease could also be induced in genetically susceptible mice by immunization with porcine cardiac myosin (PCM). The cardiac lesions induced by both antigens were characterized by extensive infiltration of the myocardium accompanied by myocyte necrosis. A novel finding was the presence of multinucleated giant cells and eosinophils in the cardiac infiltrates, in addition to a mixture of mononuclear cells and polymorphonuclear cells described previously. Immunohistochemical staining demonstrated that the mononuclear cells consisted predominantly of macrophages, CD4+ T cells and, to a lesser extent, CD8+ T cells and B cells. In addition, increased cardiac expression of adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1) were demonstrated in mice that developed myocarditis as compared with those that did not develop disease upon immunization with either PCM or MCM. The levels of TNFalpha detected in spleen cell culture supernatant were found to be higher in mice that developed myocarditis than in those that did not develop the disease. Mice immunized with PCM generated T cells and B cells reactive not only with PCM but also with MCM, and vice versa. In addition, the serum levels of IgG1 anti-MCM antibodies produced in mice immunized with PCM as well as MCM were found to correlate positively with the development of myocarditis. Such a detailed characterization of the murine model of autoimmune myocarditis induced by PCM or MCM allowed us to compare the disease process induced by homologous self and foreign antigens.