[Modern approach to early diagnosis and pathogenetic treatment of diabetic retinopathy]. / Sovremennye podkhody k rannei diagnostike, patogeneticheskomu lecheniyu diabeticheskoi retinopatii.

AIM: To improve the approach to pathogenetic treatment of diabetic retinopathy (DR) through early diagnosis and a new method for predicting disease progression. MATERIAL AND METHODS: The study enrolled 330 type 2 diabetes patients with DR (660 eyes), of whom women constituted 64.6%, men - 35.4%. The mean patient age was 62.3±2.3 years. Three groups were formed: the controls - 30 healthy volunteers (60 eyes) and 30 type 2 diabetes patients without ocular involvement (DR 0, 60 eyes); group 1 - 30 type 2 diabetes patients with DR I but no diabetic macular edema (DR I without DME, 60 eyes) that were treated with calcium dobesilate; group 2 - 240 type 2 diabetes patients, who had diabetic retinopathy of different stages (DR I, II, or III with DME, 480 eyes) and received laser retinal photocoagulation (LRP). The groups were all alike in terms of sex and age distribution. All patients underwent ophthalmic examination, including best corrected visual acuity (BCVA) and critical flicker fusion frequency (CFFF) testing, tonometry, biomicroscopy, MAIA fundus microperimetry, optical coherence tomography (OCT), and fluorescein angiography (FAG) of the retina. Traditionally we also determined blood sugar and glycated hemoglobin levels as well as vascular endothelial growth factor (VEGF-A) and monocyte chemoattractant protein (MCP-1) in tear fluid by ELISA. RESULTS: In group 1, which was under conservative therapy with calcium dobesilate, there was an increase in BCVA by the average of 0.95±0.02 and CFFF by 42.5±0.2 Hz (p<0,05). The mean central retinal thickness decreased reliably down to 265.1±12.1 µm (p<0.05). Light sensitivity of the macula improved and scored 24.13±12.3 dB (p<0.05). In group 2, the mean central retinal thickness appeared to be 383.1±221 µm, which was reliably higher than that in healthy individuals (p<0.05) and in type 2 diabetes patients without diabetic retinopathy (DR 0) (p<0.05). Tear assessment 12 months after the treatment revealed a significant decrease in VEGF-A and MCP-1 concentrations - down to 655.1±86.1 pg/ml and 1133 pg/ml, respectively (p<0.05). CONCLUSION: Conservative treatment with calcium dobesilate has proved effective in patients with DR I without DME as it ensures improvement and stabilization of the state of the retina (clinical and morphological) in one month already (judging from FAG and OCT findings). Laser treatment is rational in DR I, DR II, and DR III patients, whose condition is complicated with DME. Improvement and stabilization take, however, longer to be achieved - up to 1 year (according to FAG and OCT). Tear fluid assessment for particular participants in disease pathogenesis, such as VEGF-A and MCP-1, is a unique method for disease control and patient follow-up with account to different treatments. A new method for predicting the progression of diabetic retinopathy and diabetic macular edema has been suggested (RF patent for invention №2520826).

[Results of the use of antioxidant and angioprotective agents in type 2 diabetes patients with diabetic retinopathy and age-related macular degeneration].

AIM: to investigate changes in clinical, functional, and morphological parameters of the retina in type 2 diabetes patients with diabetic retinopathy (DR) and those with combined fundus pathology (DR plus age-related macular degeneration (AMD)) before and after a course of antioxidants and angioprotectors in the form of mono- or combination therapy. MATERIAL AND METHODS: The study included 180 patients (180 eyes) with type 2 diabetes divided into 6 groups of 30 each. DR was graded according to E. Kohner and M. Porta classification, AMD--AREDS classification. Thus, group 1 consisted of patients with DRO,; group 2--DR1 without DM, group 3--DR1 with DM, group 4--DRO and "dry" AMD (AREDS 1-3), group 5--DR1 with no DM but with AMD (AREDS 1-3), and group 6--DR1 with DM and AMD (AREDS 1-3). A drug containing lutein 6 mg, zeaxanthin 0.5 mg, vitamin C 60 mg, vitamin E 7 mg, vitamin A 1.5 mg, vitamin B2 1.2 mg, rutin 25 mg, zinc 5 mg, selenium 25 mcg, and bilberry extract 60 mg was used for antioxidative therapy. Ginkgo biloba leaf extract 60 mg was chosen as the angioprotector. In all patients visual acuity, macular thickness and morphology (OCT) as well as light sensitivity (microperimetry) were assessed before and after the treatment course. RESULTS: Analysis of baseline measurements demonstrated a significant decrease in best corrected visual acuity (p < 0.05) in study groups 2-6 as compared with group 1. Macular thickness was increased in all groups, however, the changes were statistically significant only in groups 3 and 6 (p<0.05). Light sensitivity of the macula showed a reduction, which was statistically significant in groups 4-6 (p < 0.05). After the course of antioxidant and angioprotective therapy, these parameters improved in all groups. The greatest effect was achieved with simultaneous antioxidant and double-dose angioprotective therapy (240 mg per day): visual acuity increased significantly (p < 0.05) in all groups except group 1; macular thickness decreased in all groups, however, the changes were statistically significant (p < 0.05) only in groups 1-3 and 5; light sensitivity also improved in all groups, significantly (p < 0.05) in groups 1-3 and 4. CONCLUSIONS: Extended analysis of clinical, functional and morphological changes in the retina at the onset of DR in type 2 diabetes patients with concomitant "dry" AMD enables timely diagnosis, prognosis, prevention, and early treatment. Conservative treatment with antioxidant and angioprotective agents has been proved effective in type 2 diabetes patients with preclinical (DRO) and early (DR1) diabetic retinopathy and those with DR and "dry" AMD (AREDS 1-3) in terms of functional and morphological parameters of the retina. From all the regimens, a combined antioxidant and double-dose angioprotective (240 mg) therapy appeared to be the most effective and can be considered not only a preventive, but also a therapeutic measure in type 2 diabetes patients with initial stages of DR (DRO, DR1) or those with DR and DM or combined DR and AMD (AREDS 1-3).

[Current data on the role of anthocyanosides and flavonoids in the treatment of eye diseases].

Anthocyanins are known to have antioxidant, anti-inflammatory, neuroprotective, and anticarcinogenic activity as well as positive effect on the cardiovascular system. Because of bilberry anthocyanosides (Vaccinium myrtillus L.), Mirtilene forte promotes rhodopsin synthesis and regeneration, increases retinal sensitivity to changes in light intensity, improves visual acuity and dark adaptation as well as blood supply of the retina. Studies conducted in Russia are aimed at evaluating the use of Mirtilene forte in age-related macular degeneration, diabetic retinopathy, primary open-angle glaucoma, and other diseases. This article provides an analysis of foreign and Russian publications on the effects of anthocyanins and flavonoids in different diseases.

[The effect of fibrinolytic therapy for proliferative diabetic retinopathy complications on the angiogenic potential of the eye].

OBJECTIVE: To investigate dynamic changes of pathogenically significant factors of tear fluid in type 2 diabetes patients with hemorrhagic proliferative diabetic retinopathy. MATERIAL AND METHODS: A total of 30 patients (30 eyes) with type 2 diabetes mellitus and proliferative diabetic retinopathy complicated by preretinal and vitreous hemorrhages were assessed and treated with recombinant prourokinase (Gemaza). Levels of VEGF A, MCP-1, MMP-2, and MMP-9 in tear fluid were also measured. RESULTS: Intravitreal injections of recombinant prourokinase led to more considerable gain of visual acuity and decrease of vitreous hemorrhage index in the first 24 hours in comparison with other treatment schemes. Changes of VEGF A and MCP-1 tear levels were insignificant. Increase of matrix proteinases activity was mainly due to MMP-9. CONCLUSION: The use of recombinant prourokinase is associated with clinical improvement in patients with hemorrhagic diabetic retinopathy causing no notable changes in tear levels of proangiogenic cytokines and matrix proteinases.

[Pathogenic effect of pandemic influenza virus H1N1 under replication in cultures of human cells].

The propagation of the pandemic influenza virus H1N1 in cultures of bronchial (Calu-3) and intestinal (Caco-2) differentiated epithelial cells of human origin was studied. The canine epithelial cell lines, MDCK-H and MDCK-2, were comparatively tested. The two human cell lines were found to be highly sensitive to the influenza pandemic strains A/Hamburg/05/09 and A/Moscow/501/2011 and maintained their replication without addition of trypsin to culture medium. Virus strains of seasonal influenza H1N1, such as A/Moscow/450/2003, A/Memphis/14/96, and laboratory strain A/PR/8/34, multiplied in these human cells in similar manner. The intracellular cleavage HA0-->HA1+HA2 by the host virus-activating protease (IAP) occurred in both human cell lines under infection with each influenza virus H1N1 including pandemic ones. Comparatively, this cleavage of all influenza H1N1 virus strains appeared to be either undetectable or low-detectible in MDCK-H and MDCK-2, respectively, thereby implying low levels of active IAP in these cells. Multiplication of pandemic and seasonal influenza H1N1 viruses in Calu-3 and Caco-2 cells caused cytopathic effect, which was accompanied with low autophagy and apoptosis events. These data allow recommending human cell lines, Calu-3 and Caco-2, for optimized isolation and passaging of clinical strains of Influenza pandemic viruses H1N1.

[On some pathogenic features of diabetic retinopathy in type II diabetes mellitus and the role of antioxidants and ginkgo biloba].

Diabetic retinopathy is a microvascular complication of diabetes and has complex and multifactorial pathogenesis. Cascade of biochemical changes characteristic to other diabetes vascular complications leads to structural changes of retinal capillaries some of which are unique. As a result of all processes at different pathogenesis levels there is an increase of free radicals concentration and decrease of antioxidant protection thus provoking an oxidative stress in retina and endothelial dysfunction with subsequent hypoxia and activation of growth factors and promotion of neovascularization leading to loss of vision in patients with type II diabetes mellitus. Consideration of oxidative stress reduction and restoration of retinal antioxidant system using exogenous antioxidants is a promising issue for further research.

[Results of antioxidant therapy for non-proliferative diabetic retinopathy in patients with type 2 diabetes mellitus].

Diabetes mellitus is associated with dangerous vascular complications, including diabetic retinopathy (DR), which can lead to complete visionloss. Modern diagnostic methods, such as optical coherent tomography, help accurately and objectively verify the stage of DR, thus providing an opportunity to choose a treatment algorithm able to prevent further progression of the disease. The aim of the study was to investigate the efficacy of antioxidant therapy (Vitrum Vision Forte and Vitrum Memory) as the initial phase of treatment of non-proliferative DR in type 2 diabetes, what is pathogenically reasonable due to involved oxidative stress.

[Kallikrein-kinin system as a target for diabetic retinopathy treatment].

Multifactor etiology of diabetic retinopathy (DR) determines difficulty of understanding of pathogenesis and need of search of effective approaches to study key mechanisms of development of this microvascular complication of diabetes mellitus (DM). Significant achievements of the last years show the contribution of two proteolytic systems into pathogenesis of DR, that control vascular tone and permeability - kallikrein-kinin (KKS) and renin-angiotensin systems (RAS). Among new approaches to DR treatment one of the most appropriate is an influence on KKS by means of inhibiting kallikrein, that leads to reduction of retinal vascular permeability and allows to prevent the development of macula oedema and other consequences of vascular wall damage in DR.

[Evolutionary changes in the NA and HA genes of H3N2 influenza virus in the Moscow Region during 2003-2009].

During the winter 2009 outbreak in the Moscow Region, H3N2 influenza viruses were isolated from the nasopharyngeal washes of patients via their propagation in the human intestinal (Caco-2) and bronchial (Calu-3) epithelial cell cultures maintaining the proteolytic cleavage of HA0--> HA1+HA2 and multicycle virus replication. Analysis of the nucleotide sequences of virus RNA indicated that the 2009 viruses differed from those isolated in 2003 in 14 and 21 amino acids of the neuraminidase (NA) and hemagglutinin (HA) genes, respectively. The NA gene was 1762 nucleotides long whereas the 2003 isolates had a deletion of 66 nucleotides (22 amino acids) in the stalk region (short-stalk NA genotype) of viruses. The NA gene of the 2009 and 2003 isolates possessed an amino acid profile characterized for oseltamivir- and zanamivir-susceptible viral strains. The HA gene of the 2009 viruses contained an N-glycosylation site at Asn181 (an analog to Asn 65 numbering from a signal peptide), which correlated with the long-stalk NA gene. The 2009 viruses had Phe209 in the HA receptor binding center whereas the 2003 isolates possessed Ser209, which correlated with their differences in HA activity. Phylogenetic analysis showed that the NA genes of the 2003 and 2009 Moscow strains were located in the same genetic clade with a single common precursor while their HA genes were diverged in more genetic distance and located in different clades. Viral distribution in the phylogenetic tree indicated that the Moscow strains isolated in 2009 were not direct ancestors of those isolated in 2003; and during the period of 2003 to 2009, H3N2 influenza virus with a short-stalk NA genotype was substituted for a migrant virus possessing a long-stalk NA gene.

[Site-directed modification of caspase cleavage site regions in avian influenza virus proteins].

A reverse genetics approach was applied to generate variants of avian influenza virus A/FPV/Ro/34 (H7N1) containing mutations in the caspase cleavage sites of NP and M2 proteins. Mutation Gly16 --> Asp in avian virus NP made this protein (NPgd) sensitive to caspases, like human virus NP, and permitted its cleavage in infected cells. Mutant recombinant virus NPgd was able to replicate and stably carried Gly --> Asp mutation during passages in cultured cells, chicken eggs, and chickens. This variant was found to have significantly decreased virulence for chickens comparatively to wild type recombinant virus (wtr). Virus variants characterized by deletion Gly16 in NP (NPdel) and mutated caspase cleavage site VDVDD87 --> VNVND87 in M2 (M2nn) protein were shown to lack intracellular caspase-dependent cleavage of NP and M2, respectively, and to retain their ability to replicate in different hosts. Variant NPdel, like wide type virus, displayed a high chicken virulence whereas M2nn, like NPgd one, was found to possess a low virulent phenotype. The findings suggest that the mutations altering natural caspase cleavage motifs in NP and M2 do not restrict virus replication ability but can significantly reduce the virulent potential of the mutant viruses. Recombinant virus variants with altered caspase cleavage motifs could be proposed as a matrix for the design of live recombinant vaccines.

[Key role of Asp16 in proteolysis of influenza A NP protein by caspases in infected cells]. / Kliuchevaia rol' Asp16 v proteolize belka NP virusa grippa A kaspazami v infitsirovannykh kletkakh.

The main nucleocapsid protein NP (molecular weight--56 kD) of human influenza A virus (IAV) was found to be subject to the N-terminal proteolysis in position Asp16 with production of aNP (molecular weight--53 kD) in the infected cells' apoptosis. It was assumed that NP of avian and animal influenza viruses was not subject to proteolysis since it has Gly16. To verify the above assumption the NP chimeric gene of human influenza virus was developed; Asp16 was replaced by Gly by means of "site-oriented" mutagenesis in the above gene, after that, the A/WSN/33 (H1N1) mutant of human influenza virus with "avian" NP and with point mutation (Gly16) was developed by using the method of "reverse genetics". The "human" influenza virus with "avian" chimeric NP/Gly16 turned out to be viable but had a lower replication velocity versus its wild-nature counterpart. It is noteworthy, that the mutant virus caused the cellular apoptosis in the remote infection period the way the wild virus did; however, NP of the former was found to be resistant to cellular caspasas and was not subject to proteolysis in infected cells. The conclusion is that Asp16 in NP molecule of human IAV is involved into the regulation process of virus replication and is the key element in NP proteolysis by cellular caspasas in cells' apoptosis.