Mechanisms of immune regulation and transplantation immunity in corneal transplants

At the present time, corneal transplantation (keratoplasty) is one of the most frequent modes of solid tissue transplants in the world. Unlike other kinds of transplants, corneal grafting is often performed without tissue typing and systemic immunosuppression.High frequency of transparent corneal engraftment (up to 90% of cases) in the absence of risk factors is due to special immunoprivileged area in the anterior eye segment (functionally, a structural aggregation of the cornea and anterior chamber, AC) accomplished by local and systemic immunoregulatory mechanisms, i.e., phenomenon of immune deviation associated with anterior chamber of the eye (ACAID), components of the internal liquid medium, a watery moisture with immunosuppressive properties, e.g., IL-1ra, TSP-1,TGF-β2, regulatory complement proteins, α-MSH (alpha-melanocyte stimulating hormone), VIP (vasoactive intestinal peptide), indolamine 2,3-dioxygenase (IDO), calcitonin-gene-bound peptide (CGRP), somatostatin, etc.In addition to ACAID and liquid AC components, a contribution to the maintenance of immune privilege which is extremely important for a successful outcome of keratoplasty, is provided by other mechanisms, in particular, immunologically active membrane-associated molecules of corneal endothelium, i.e., PDL-1 (Programmed death ligand 1), and sVEGFR-1, sVEGFR-2, sVEGFR-3 involved in maintaining avascularity of the corneal tissue. Disturbances of the immune privilege of the cornea promotes activation of immune recognition with switching the effector mechanisms of transplantation immunity, thus leading to subsequent development of the tissue incompatibility reaction and clouding of transplanted cornea. Graft rejection can be localized in any of the corneal cell layers, including epithelium, stroma, and endothelium. Endothelial rejection causes the most severe affection of visual functions, due to the inability of local endothelial recovery, and water accumulation due to the endothelial dysfunction.Graft rejection is clinically characterized by edema and the presence of inflammatory cells, either circulating in the anterior chamber, or forming precipitates on the graft endothelial cells.A number of factors are associated with an increased risk of corneal graft rejection, including the degree of inflammation and/or vascularization of the transplant bed i.e., location of the donor cornea, repeated keratoplasty, allosensitization due to other cellular transplants, including bone marrow, blood transfusions, pregnancy, etc., as well as allergic and systemic diseases.This review article considers and systematizes the data from the literature concerning studies of the factors determining the immune privileged state of cornea, and the ACAID phenomenon, their role in development of allotolerance in corneal transplantation, highlights the main conditions required for triggering the tissue incompatibility reactions, discusses the mechanisms of allogeneic recognition and effector stage of the immune response, destruction of corneal allografts

Local and systemic production of 45 cytokines in complicated proliferative diabetic retinopaty

Diabetic retinopathy (DR) is multifactorial by its origin, involving many cytokines and growth factors. Studies of cytokine levels in biological fluids seem to be relevant for an in-depth understanding of the disease pathogenesis. The purpose of the work was a comparative analysis of 45 cytokines at systemic (blood serum (BS)) and local (vitreous humor (VH)) levels in the patients with complicated proliferative DR, showing various features of the clinical pattern. The content of cytokines was tested in 53 samples of BS and 32 samples of VH in 53 patients with type 1 and type 2 diabetes mellitus with severe proliferative DR. We used the multiplex analysis technique by means of xMAP platform and Luminex xPONENT 3.1 program using 45-plex sets (Procarta Plex «eBioscience», Austria). 25 cytokines were detected at significant amounts in BS test samples, and 27 cytokines were revealed in VH specimens. Sensitivity limits of the test system allowed to find significantly higher levels of 7 cytokines (IL-6, IL-8, IP-10, MCP-1, HGF, LIF and VEGF-A) in VH samples, than in the BS, thus indicating to their local intraocular production. Correlations between the contents of VEGF-A growth factor and amounts of cytokines, including those involved in inflammatory reactions, are shown in VH, thus presuming the interrelation of pathogenetic components, i.e., inflammation and neoangiogenesis. The features of intraocular cytokine content were determined for various manifestations of diabetic ocular changes. Hemophthalmus has been shown to be associated with increased IL-8 and IP-10; iris rubeosis, with increase in LIF; proliferative DR activity was associated with higher MCP-1 levels, and extremely severe changes were related to increase in IL-6 and EGF. Testing of cytokines in biological fluids is informative when studying the mechanisms of inflammation, neoangiogenesis, and protective responses in pathogenesis of diabetic retinopathy

Proven and less studied hematopoietic and vasoactive growth factors in retinal capillary hemangioma

Pathogenesis of retinal capillary hemangioma has not been sufficiently studied at the present time. Therefore, the study of cytokine levels in biological fluids seems to be very relevant in order to increase knowledge about the mechanisms of the disease development and searching for targeted therapies. The content of hematopoietic and vasoactive growth factors in blood serum, lacrimal fluid, and vitreous body was studied in patients with retinal capillary hemangioma. A total of 26 patients with retinal angiomatosis were examined. The samples of blood serum (n = 23) and lacrimal fluid (n = 10) from practically healthy people aged 22 to 46 (27.4±1.4 years) were used as a control. To perform comparative assessment of cytokine concentrations in the vitreous body of patients with retinal capillary hemangioma, were used samples of the vitreous body from 6 patients (average age 33±4.7 years; from 21 to 49 years) with rhegmatogenous retinal detachment. To measure the cytokine concentrations, we applied multiplex analysis technique using the xMAP platform with LuminexxPONENT 3.1 program and ProcartaPlex sets (eBioscience, Austria). A detailed characteristic of vasoactive factors in capillary retinal hemangioma was obtained as a result of this work. Some disorders in chemokine regulation were identified. There was a significant increase in serum concentrations of three vasoactive factors, i.e., PDGF-BB, HGF, and PIGF-1, with a decrease in chemokines (MCP-1, MIP-1α, and MIP-1β). The frequencies of PIGF-1 and MIP-1α detection also significantly differed from the control group. SCF was significantly more often determined in patients with retinal angiomatosis only at the systemic level. Correlations between PDGF-BB and PIGF-1, as well as PIGF-1 and MIP-1β were shown. A significant increase in VEGF-A, HGF, VEGF-D, as well as MCP-1 concentrations was shown in the lacrimal fluid. The inversion of PDGF-BB concentrations in serum and lacrimal fluid was noted. Analysis of intraocular cytokine levels revealed a significant increase in VEGF-A and HGF concentrations, with marked decrease in MIP-1α and MIP-1β. PDGF-BB in 100% of cases was determined only in vitreous body of patients with retinal angiomatosis. With respect to the revealed characteristic shifts of HGF/SF intraocular production in retinal capillary hemangioma, it seems relevant to search ways for its inhibition, thus providing potential basis for a new therapeutic strategy in treatment of retinal angiomatosis

Screening of cytokines in blood serum and lacrimal liquid in wet and atrophic forms of age-related macular degeneration

Cytokines play an integral role in pathogenesis of age-related macular degeneration (AMD). Of particular interest are the late stages of this disease, which causes progressive visual impairment. Therapy-induced effects of post-treatment cytokine concentrations also need to be studied, both at long and short observation terms. These studies are of vital importance if the atrophy occurs during antiangiogenic therapy. Our purpose was to study an array of 45 cytokines, in blood serum (BS) and lacrimal liquid (LL) of the patients with wet and atrophic AMD.The study included 70 people (85 eyes) with stage 3-4 AMD according to AREDS. Depending on the form of AMD, 3 groups were discerned: I group (n = 24) included the patients with “geographic atrophy”; II group (n = 22), consisted of the patients with macular atrophy treated with antiangiogenic therapy of wet AMD; III group (n = 24), comprised the patients with a wet AMD who did not previously receive the treatment. Control group consisted of healthy volunteers (n = 25). All the groups were comparable for age and gender. The patients underwent a comprehensive ophthalmological examination to make a diagnosis. A multiplex study of the local (in the BS) and systemic (in the LL) cytokine status was carried out on a MAGPIX device (platform хMAP, Luminex Corporation, USA) in the Luminexx PONENT 3.1 software, using Procarta Plex kits (eBioscience, Austria). We determined 45 cytokines causing various biological effects, i.e., IL-1á, IL-1â, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, IFNá, IFNã, IL-8/CXCL8, IP-10/CXCL10, SDF-1á/CXCL12, MCP-1/CCL2, MIP-1á/CCL3, MIP-1â/ CCL4, RANTES/CCL5, Eotaxin/CCL11, TNFá, TNFâ, GM-CSF, VEGF-A, VEGF-D, FGF-2, EGF, PDGF-BB, HGF, SCF, GRO-á, NGF-â, BDNF, LIF, PIGF-1.Screening of a wide range of cytokines showing various biological effects was carried out in BS and LL of patients with atrophic and wet forms of AMD. It has been shown that the late stages of the disease are associated with local and systemic changes of pro / anti-inflammatory mediators (IL-1â, IL-1ra, IL-18, LIF), chemoattractant cytokines (IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, MIP-1á/CCL3, MIP-1â/ CCL4, RANTES/CCL5, Eotaxin/CCL11), hematopoietic regulators (IL-7), and growth factors with known angiogenic activity (EGF, HGF, PDGF-BB, VEGF-A). Altered concentrations of numerous chemokines, e.g., IP-10/CXCL10, SDF-1á/CXCL12, MIP-1á/CCL3, MIP-1â/CCL4, RANTES/CCL5 and Eotaxin/CCL11 (p < 0.05) in BS of the patients with atrophic and wet AMD may be of interest for the search of biomarkers associated with various clinical phenotypes of the disease and may be also helpful for development of new therapeutic strategies

Investigation of local expression of NLRP3 inflammasome complex genes in modeling retinal degeneration <i>in vivo</i>

Neurodegenerative ophthalmopathology is one of the main causes of irreversible blindness and disability in the world. In the pathogenesis of diseases of this group, more and more attention has recently been paid to the role of local inflammation caused by the activation of innate immunity and the mechanisms of its genetic regulation. In recent years, works have appeared in the field of experimental ophthalmology that have demonstrated the possibility of NLRP1, NLRP3 inflammasome complexes assembling when exposed to hyperglycemia, oxygen deprivation of retinal cells, as well as modeling compressive stress similar to that in glaucoma [15]. However, the mechanism of inflammasome involvement in the development of neurodegenerative eye diseases remains unclear. The aim of the study was to investigate the local expression of genes encoding proteins of the NLRP3 inflammasome complex (NLRP3, CASP-1) in an experimental model of retinal degeneration in rabbits. The studies were performed on samples of tissue complex (TC) of the retina/retinal pigment epithelium (RPE) (retina/RPE TC), isolated from the eyes of 14 New Zealand albino rabbits, in which degenerative retinal lesion was modeled by a single subretinal injection of 0.01 mL of 0.9% sodium chloride solution, and 7 healthy rabbits without eye damage. The formation of retinal degeneration was judged on the basis of changes in morphofunctional parameters obtained during specialized ophthalmological research methods (optical coherence tomography, fundus autofluorescence, electroretinography) at follow-up periods of 1, 3 and 6 months. The level of expression of NLRP3 and CASP-1 genes in the retina/RPE TC was evaluated by reverse transcription polymerase chain reaction (RT-PCR). According to the results of the study, a statistically significant increase in NLRP3 gene expression (p &lt; 0.001) was noted in the retina/RPE TC of experimental animals, which may indicate the involvement of NLRP-3 inflammasome components in the development of neurodegenerative retinal lesions. At the same time, the expression of the gene encoding CASP-1 was detected only in the retina/RPE TC of experimental eyes and is probably due to local inflammatory mechanisms in the retinal tissue.The high level of NLRP3, CASP-1 mRNA, detected in all retina/RPE TC samples of experimental eyes at late stages of the experiment (3 and 6 months), allows us to assume the formation of mechanisms (for example, activated glial phenotype) that support inflammation in retinal tissue. This should be taken into account in actively developing transplantation methods for the treatment of retinal degeneration

EXAMINING LOCALLY EXPRESSED mRNA OF INFLAMMATORY MEDIATOR GENES IN A MODEL OF RETINAL PIGMENT EPITHELIUM ATROPHY AND RETINAL DEGENERATION INDUCED BY SUBRETINAL SALINE INJECTION IN RABBITS

Degenerative-dystrophic retinal diseases, particularly age-related macular degeneration (AMD), are now considered to be the lead cause of blindness and low vision in developed countries, with a steadily increasing trend. Recent publications provide evidence for the involvement of inflammatory mechanisms in TMD development and progression unveiled due to advances in innate and adaptive immunity research. However, the immunopathogenesis of atrophic AMD form, “geographic atrophy” (GA) remains largely unstudied. Objective: to investigate local mRNA expression of inflammatory cytokines IL-1β, IL-18, CCL2/MCP-1 in a model of RPE atrophy induced after subretinal injection of 0.9% sodium chloride solution in experimental rabbits. The investigation was carried out in tissue complex retina-RPE-choroid (TC) samples isolated from eyes of 23 albino New Zealand rabbits after modeling RPE atrophy by subretinal injection of 0.9% sodium chloride solution and 5 healthy rabbits lacking eye lesions. Animals in the experimental group (one week before surgical intervention, in the early period, and in the period of sustained RPE atrophy formation) and controls were subjected to optical coherence tomography (OCT) and ocular fundus autofluorescence (FAF). Evaluation of proinflammatory cytokine gene expression levels in TC was performed by RT-PCR. Results. Subretinal injection of 0.01 ml of 0.9% sodium chloride solution induced experimental RPE atrophy development in rabbits vs. control that was associated with multidirectional changes of IL-1β, IL-18, MCP-1/CCL2 gene mRNA expression. Three types of response in the TC, formed during development of atrophic changes and determined by the value of local cytokine gene expression were characterized: 1) hypo/ no response – decreased/no expression; 2) normal response – moderate increase; 3) hyper response – overexpression. 69.6% of animals with persistent atrophy had a moderate to hypertrophic increase in locally expressed mRNA MCP-1/CCL2, whereas 30% cases had significantly increased IL-1β mRNA expression – factors damaging the blood-retinal barrier and contributing to posterior segment immune privilege. It should be taken into account while developing new strategies for treatment of ophthalmic pathology, in particular the currently actively studied and tested options for RPE stem cell transplantation into subretinal space. The data obtained may be useful to investigate various types of RPE atrophy and develop new strategies of ophthalmopathology treatment in preclinical studies

Липосомы, содержащие дексаметазон: получение, характеристика и использование в офтальмологии

Glucocorticoid dexamethasone (DM) liposomes of various lipid compositions were obtained. DM was used in insoluble form and in water-soluble form as disodium salt of dexamethasone phosphate (DMP). Correspondence between the drug inclusion percent and the structure of liposomes, and its solubility in water was studied. Lipids were shown to allow DM solubilization at concentration exceeding its solubility in water 9.3 times. Using repeated gel-chromatography, release of medicinal substances from liposomes was determined for all the samples of liposomes containing both DМP and DM. It was thus shown that up to 87% of the included substance remained in the liposomes. Biocompatibility of «empty» liposomes and liposomes containing a glucocorticoid with rabbit eyeball tissues was studied after their endovitreal incorporation. Dipalmitoylphosphatidylcholine liposomes were demonstrated not to cause inflammatory reaction after introduction in this way. Distribution of the liposomes in tissues of the rabbit eye posterior chamber was determined. It was shown that the liposomes after endovitreal introduction were distributed in various layers of retina.Получены липосомы различного липидного состава, содержащие глюкокортикоид дексаметазон (ДМ) и динатриевую соль дексаметазона фосфата (ДМФ). Изучена зависимость эффективности включения препарата от его растворимости в воде и от состава липосом. Показано, что липиды позволяют солюбилизировать ДМ в концентрации, в 9.3 раза превышающей его растворимость в воде. Для всех образцов липосом, содержащих как ДМФ, так и ДМ, определена степень высвобождения лекарственных субстанций из липосом путем проведения повторной гель-хроматографии. При этом показано, что в липосомах сохраняется до 87% включенной субстанции. Изучена биосовместимость «пустых» липосом и липосом, содержащих глюкокортикоид, с тканями глазного яблока кролика при их эндовитреальном введении. Установлено, что липосомы из дипальмитоилфосфатидилхолина не вызывают воспалительной реакции при таком способе введения. Изучено распределение липосом в тканях заднего отдела глаза кролика. При этом показано, что липосомы при эндовитреальном введении распределяются в различные слои сетчатки