Reaction of homopyrimidine oligonucleotides bearing a 5'-terminal alkylating aromatic 2-chloroethyl-amino group with a bovine papilloma vector expressing human interferon-gamma was investigated. The oligonucleotide derivatives bound to corresponding homopurine-homopyrimidine sequences in dsDNA and alkylated guanosine residues at these sites in the purine strand of the target. The alkylated DNA can be cleaved at the modified residues. At pH 5.4, the reaction was highly specific to the target sequences; at pH less than 5, some nonspecific reactions were observed at the sequences partially complementary to the oligonucleotides. Elongation of the linker between the alkylating group and the oligonucleotide phosphate increased the alkylation efficiency. Repeated treatment of the DNA with gradually increased concentrations of the reagent resulted in quantitative modification of the target guanosines.

We used a DNA duplex formed between the 5' end of a 69mer (69T) and an 11mer (OL7) as a substrate for BamHI. The former oligonucleotide folds into a hairpin structure, the stem of which contains a stretch of pyrimidines in one strand and consequently a stretch of purines in the other strand. The oligomer 69T was used as a target for complementary oligodeoxypyrimidines made of 10 nt (OL1), 16 nt (OL5) or 26 nt (OL2) which can engage the same 10 pyrimidine-purine-pyrimidine triplets with the 69T hairpin stem. Although the binding site of OL7 did not overlap that of OL1, OL2 or OL5, the BamHI activity on 69T-OL7 complexes was drastically modified in the presence of these triplex-forming oligomers: OL1 abolished the cleavage by BamHI whereas OL5 and OL2 strongly increased it. Using footprinting assays and point-mutated oligonucleotides we demonstrated that these variations were due to different conformations of the 69T-OL7 complex induced by the binding of oligomers OL1, OL2 or OL5. Therefore, oligonucleotides can act as structural switchers, offering one additional mode for modulating gene expression.

Sequence-specific alkylation of dsDNA with pyrimidine oligonucleotides bearing an alkylating group at the 3' and 5' terminal phosphates, or both, has been investigated. At pH 5.4, sequence-specific modification of guanosines of the DNA in the vicinity of the target purine-pyrimidine sequence occurs. The reactive group at the 5' terminus of the oligonucleotides attacks guanosines in the purine strand of the target DNA. The reactive group at the 3' end can interact with guanosines in both strands of the DNA. Bifunctional reagents can alkylate both strands of the DNA simultaneously. At pH 4 in the presence of magnesium ions, the oligonucleotide derivatives can form imperfect complexes with sequences homologous to the target sequence and alkylate the DNA at the corresponding positions.

The clinical course of active retinopathy after anti-VEGF therapy, the possibility and timing of recurrence of the disease, anatomical and functional outcomes of treatment are widely discussed in the press, not fully studied and relevant.Purpose: to study the clinical course of active retinopathy of prematurity after anti-VEGF therapy and clinical and functional outcomes.Patients and Methods. Children with active retinopathy of prematurity, who turned to the Helmgoltz National Medical Research Centre of Eye Diseases after anti-VEGF therapy, examined by indirect binocular ophthalmoscopy and digital retinal camera (RetcamShuttle). Children were monitored from 1.5 to 6 years (average 2.94 ± 1.47). All children underwent routine examination, 4 children older than 3 years underwent optical coherence tomography.Results. In all cases, after anti-VEGF therapy, there was a decrease in vascular activity and continued vascularization of the retina. Recurrence of the disease requiring additional treatment, were detected in 11 (42.3 %) eyes within 6–22 weeks (in average 13.33 ± 5.57) after intravitreal anti-VEGF therapy. Laser coagulation of the retina was carried out in 4 children (7 eyes) and repeated administration of anti-VEGF drug — 2 children (4 eyes), which led to regression of the disease. In the long-term period, all 13 (100 %) children had successful outcomes.Conclusion. Anti-VEGF therapy is effective in plus-zone 1 disease and posterior aggressive retinopathy of prematurity. Its advantages include the ability to treat retinopathy in zone 1 posterior, the absence of “blockade” of the peripheral retina with the possibility of continued growth of blood vessels to the periphery, lower frequency and severity of myopia. The disadvantages include the possibility of recurrence of the disease, which requires long-term regular monitoring.

Insufficient effectiveness of laser coagulation of the avascular retinal areas in retinopathy of prematurity (ROP) plus-disease in zone I and aggressive posterior retinopathy of prematurity (APROP) requires new treatment approaches, based on the regulation of retinal angiogenesis and anti-VEGF drugs use. The BEAT-RAP study, which was the first major randomized study of anti-VEGF therapy in ROP, revealed a higher effectiveness of bevacizumab compared to retinal laser coagulation in stage 3 plus-disease of zone I. A prospective randomized trial, RAINBOW, demonstrated the effectiveness of ranibizumab in plus-disease stages 1, 2 and 3 in zone I and stage 3 in zone II and in APROP, so that the drug may be recommended for use in children with ROP. The demonstrated high effect of anti-VEGF therapy in ROP is consistent with our own data. Anti-VEGF therapy opens up new possibilities in the treatment of a particular class of ROP forms. The advantages of anti-VEGF therapy include higher clinical effectiveness of treatment of ROP type I with localization in the posterior pole (I and posterior II zone), absence of "blockage" of the peripheral retina, lower frequency of myopia development and degree, relative fastness of the procedure, the acceptability for patients whose fundus is difficult to visualize, and somatically burdened patients who are contraindicated for prolonged anesthesia used for retinal laser coagulation. When using anti-VEGF drugs in the post-threshold stages of the disease, one should take account of an increased risk of proliferation progression and retinal detachment development. Premature infants with retinopathy regression after anti-VEGF therapy require a longer duration of regular and frequent follow-up (up to 70 weeks of postmenstrual age) due to the risk of relapse and extraretinal proliferation in future.

Background: At the development of graft versus host disease in genetically homogeneous population of (C57Bl/6 x DBA/2) F1 mice two clinical phenotypes of SLE-like disease were revealed: lupus+ (immune complex glomerulonephritis and hemolytic anemia) and lupus-(hemolytic anemia). The GvHD phenotypic heterogeneity is determined by the T h1 /T h2-polarization: T h2 lymphocyte predominant activity, leads to the lupus+ development, or prevalence activity of T h1 cells, leads to the lupus-development. Objective: Our aim was to evaluate the possibility of using an experimental model of autoimmune disease for studying and testing of epigenetic modifications, shifting T h1 /T h2balance in vivo. Methods: Сhronic GVHD was induced in B6D2F1 mice by the transplantation of 130×10 6 parental DBA/2 splenocytes. Аnti-ds-DNA, total IgG and IgG1, IgG2а Abs were measured by ELISA. Results: Six-to 8-week-old female DBA/2 and B6D2F1 mice were obtained from Biological Research Laboratory (Novosibirsk). It was established that regular moderate physical activity (unladed swimming) shifted T h1 /T h2 balance towards T h1. This leads to a decrease in a population of recipients the lupus+ mice from 57 to 26% (p <0,001) with significantly reduced hypergammaglobulinemia (IgG from 2,8 to 2,0 mg/ml; p <0,047) and DNA antibodies titer from 0,18 to 0,12 OD (p =0,05). Administration of epigenetic modificator bisphenol A at low doses, which mimicking estrogen effects, enhances the proportion of lupus+ mice in experimental groups from 33 to 64% (p <0,001) and impairs their clinical status by the increasing the urine protein level from 2.8 to 4,2 mg/ml (p <0,001) in animals. Conclusion: T h1 /T h2-balance presumably is determined by the immune system epigenetic modification in experimental mice, formed on the previous stages of ontogeny and defines the direction of immune processes development in individual animal.

The effectiveness of the in vitro fertilization (IVF) program does not exceed 40% and mostly depends on the oocytes quality, that is affected by the composition of the follicular fluid: a content of cytokines, growth factors, cell-free DNA (cfDNA), et al. Increased level of cfDNA in the follicular fluid is associated with indicators of ovarian reserve, as well as the effectiveness of stimulation in the IVF program. One possible reason for the high level of cfDNA can be considered an increase of IL-8 concentration. However, the role of IL-8 in regulation of reproductive processes is ambiguous and is presented in a few studies. In this connection, the aim of the study was to investigate a content of cfDNA and IL-8 in the follicular fluid of women, as well as the relationship between them, depending on the parameters of folliculo- and oogenesis, early embryogenesis and IVF outcomes. 62 women with infertility and undergoing IVF treatment have been enrolled in the study. We collected follicular fluid samples from dominant follicles using transvaginal ultrasound aspiration. The concentration of IL-8 in the follicular fluid was evaluated by flow fluorimetry. Measurement of cfDNA in the follicular fluid was performed by fluorimetric method. A negative correlation between IL-8 level in the follicular fluid and the number of received oocytes, as well as the quality of embryos was revealed. At the same time, a higher level of cfDNA was recorded in women with low blastocyst quality and non-developing pregnancy. Correlation analysis showed the absence of a significant direct relationship between IL-8 and cfDNA in the whole group. Moreover, in subgroups characterized by a higher IL-8 level, we found a weak negative correlation between cfDNA and IL-8 concentrations. The results suggest that enlarged level of cfDNA in the follicular fluid is not a consequence of IL-8 increase; the levels of IL-8 and cfDNA in the follicular fluid are two independent factors with multidirectional effects involved in various stages of reproductive process.

20Íîâûå àñïåêòû èììóíîïàòîãåíåçà ðåòèíîïàòèè íåäîíîøåííûõ: ðîëü TGF-1 è IGF-II â íàðóøåíèÿõ ïðîöåññà âàñêóëÿðèçàöèè ñåò÷àòêè Ë.À. Êàòàðãèíà, Î.Ñ. Ñëåïîâà , Å.Í. Äåì÷åíêî, Í.À. Îñèïîâà ФГБУ «Московский НИИ глазных болезней им. Гельмгольца» Минздрава РоссииИзучение иммунологических аспектов патогенеза ретинопатии недоношенных (РН) позволяет разрабатывать новые подходы к профилактике, рациональной диагностике и повышению эффективности лечения данного заболевания. Цель работы: анализ уровня ростовых факторов в сыворотке крови недоношенных детей группы риска развития РН на доклинической стадии и его взаимосвязи с дальнейшим развитием и характером течения заболевания. Материал и методы. Обследовано 85 недоношенных детей группы риска развития РН. Средний срок гестации при рождении детей -27,7 ± 2,2 нед, средняя масса тела при рождении -1086,1 ± 266,1 г. Обследование включало динамическую офтальмоскопию и исследование содержания в сыворотке крови VEGF-A на проточном цитометре (BD FACS Canto II), а также TGF-1, IGF-I, IGF-II методом твердофазного иммуноферментного анализа с использованием тест-систем Bender MedSystems (Австрия). Результаты. На момент первого офтальмоскопического обследования, т. е. до клинической манифестации заболевания, в сыворотке крови детей с развившейся впоследствии РН, потребовавшей проведения лазеркоагуляции аваскулярных зон сетчатки, наблюдались относительно более высокие значения концентрации VEGF-А (выше 1300 пг/мл) и IGF-II (выше 140 пг/мл) и низкие значения концентрации IGF-I (ниже 24 пг/мг) и TGF-1 (ниже 8000 пг/мл) по сравнению с группами «благополучных» детей. Динамическое исследование содержания VEGF-А одновременно с TGF-1 в процессе развития РН выявило однонаправленность изменений концентраций данных ростовых факторов на всех сроках наблюдения. Заключение. Впервые показано, что прогностически неблагоприятными в плане последующего развития тяжелой РН являются высокие значения концентрации IGF-II и низкие значения концентрации TGF-1 до клинической манифестации заболевания. Однонаправленность изменений уровней TGF-1 и VEGF-A в сыворотке крови в процессе развития РН может свидетельствовать о синергичном характере участия данных ростовых факторов в патологической вазопролиферации; детальное изучение механизма этого участия требует проведения дальнейших исследований.Ключевые слова: ретинопатия недоношенных, иммунопатогенез, TGF-1, IGF-II.