EVALUATION OF THE EFFICIENCY OF COMBINATION THERAPY WITH HYALURONATES IN PATIENTS WITH HIP OSTEOARTHROSIS

Objective: to make the results of treatment for coxarthrosis better via prosthetic synovial fluid replacement, by improving drug delivery into the joint cavity. Subjects and methods. The clinical trial enrolled 359 outpatients treated for coxarthrosis. A control group comprised 50 patients receiving the intraarticular combination drug Alflutop under ultrasound (US) guidance. A study group included 309 patients treated using a new three-stage procedure. At stage 1, the patients were given periarticular injections of the current enzyme preparation longidase with a hyaluronidase activity of 3000 IU. At stage 2, postisometric relaxation sessions were performed. At stage 3, after defining the optimal access, synovial fluid prosthetic devices were inserted into the joint cavity under US guidance (a total of 2-3 injections) once weekly. To evaluate the efficiency of the developed treatment option, the authors used a number of standard tests, WOMAC index, Leken's index, integral index of lower limb dysfunction, needs for nonsteroidal anti-inflammatory drugs; total treatment scores given by a physician and a patient; quality of life estimation using the SF-36 questionnaire. Results. The developed treatment option could significantly reduce the clinical and functional WOMAC index and Leken's index, and inadequate joint function in the immediate period and a year after the treatment performed. It also considerably improved quality of life in patients with hip osteoarthrosis, which appeared as a significant increase of its rating according to eight SF-36 scales during more than a year

Влияние мультипотентных стромальных клеток, адсорбированных на полимере молочной кислоты, на воспалительную реакцию после его экспериментальной имплантации

Цель. Изучить влияние аутологичных мезенхимальных мультипотентных стромальных клеток (ММСК) костномозгового происхождения, адсорбированных на полимере молочной кислоты - полилактиде (ПЛ), на воспалительный процесс после экспериментальной имплантации этого полимера. Материалы и методы. С помощью световой микроскопии с применением люминесценции изучали изменения подкожно-жировой клетчатки крыс после имплантации ПЛ с пассивно адсорбированными на поверхности ММСК с трансфицированным геном GFP и дополнительно окрашенными Vybrant® CM-Dil клеточными мембранами. Результаты. Спустя 1 нед рядом с имплантированным ПЛ с адсорбированными ММСК методами флуоресцентной микроскопии были найдены фибробластоподобные клетки с ярким и равномерным свечением цитоплазмы при использовании родаминового фильтра. На 2-й неделе возле ПЛ присутствовали макрофаги разных размеров и форм, с интенсивной флуоресценцией множества включений в условиях применения родаминового фильтра. Далее яркость люминесценции и количество светящихся объектов прогрессивно снижались вплоть до почти полного исчезновения к 4-й неделе. В результате адсорбции на ПЛ ММСК уменьшается объем склерозированной клетчатки с увеличением в ней числа сосудов на 1 - 2-й неделях после имплантации. В этой клетчатке в течение 1-й недели численная плотность всех лейкоцитов и лимфоцитов ниже. Типичная капсула вокруг имплантированного ПЛ у крыс формируется только к 3-й неделе. Объем капсулы, ее васкуляризация и цитограмма лейкоцитов не зависели от адсорбции ММСК. Выводы. При имплантации ПЛ с адсорбированными ММСК уже к 2-й неделе эти клеточные элементы из тканей фагоцитируются макрофагами, ММСК и их детрит полностью элиминируются из места введения к 4-й неделе. Адсорбция ММСК на ПЛ способствует уменьшению выраженности склероза и воспалительных изменений подкожно-жировой клетчатки при увеличении ее васкуляризации на 1 - 2-й неделях после имплантации. Сроки формирования капсулы вокруг ПЛ, а также ее структура не связаны с использованием клеточных технологий

Production of factors involved into fibrosis regulation by various types of human macrophages

Macrophages (Mφ) play a key role in regulation of fibrogenesis, including proliferation of fibroblasts and  myofibroblasts, differentiation of progenitor cells into  myofibroblasts, as well as synthesis  and  secretion of the  extracellular matrix, mainly  collagen.  The  direction of the  Mφ effects (stimulation or suppression)  is determined by a number of factors,  including the stage of the fibrotic process and the Mφ functional phenotype dependent on the signals of microenvironment. One  of the feasible ways of the fibrogenesis  regulating is the secretion of pro-  or antifibrotic factors such as matrix metalloproteinases, inhibitors of metalloproteinases and some cytokines. However, existing data on ability to secrete  these factors by various subpopulations of human Mφ are rare and controversial. The aim of this study was to characterize the ability of human M1,  M2a,  and M2c Mφ differentiating in the presence of GM-CSF to produce matrix metalloproteinases (MMP-9) and their tissue inhibitors (TIMP-1), as well as some cytokines and growth factors. As compared to M2 macrophages, the M1 macrophages polarized by lipopolysaccharide produced significantly  more TNFα, IL-6 and IL-2 that have pro-inflammatory activity and are able to initiate a fibrotic process.  In turn,  M2a Mφ stimulated by IL-4  were characterized by a high level of VEGF production and, at the same time, low levels of TNFα and IL-6, which may determine the important role of these cells at the proliferative stage of fibrosis and stimulation of extracellular matrix deposition. Finally, M2c Mφ polarized by dexamethasone, exhibited  the М2а-like cytokine profile, i.e., VEGF was actively produced against the background of low TNFα and IL-6 synthesis.  Moreover, all three Mφ subpopulations did actively secrete MMP-9 and TIMP-1, without significant  difference in production of these factors. However, M2c Mφ differed by a significantly  higher MMP-9/TIMP-1 ratio index compared to M1 and M2a Mφ, and it is crucial  at the rearrangement stage of the fibrotic  process.  Thus,  the production of MMP-9 and TIMP-1, together with other  pleiotropic cytokines and growth factors by various Mφ subtypes may reflect their role in regulation of fibrotic process at various stages

Effect of soluble factors of macrophages polarized by efferocytosis on neuronal density in the frontal cortex and hippocampus of mice in a model of stress-induced depression

Recently, there has been a steady increase in depressive disorders, which occupy an important place in the structure of the causes of disability. In the pathogenesis of depression, an important role is played by neuroinflammation, which is associated with impaired adult neurogenesis. Notably, neuroinflammation is partially reversible, and the leading role in the initiation and regulation of neuroregeneration is given to macrophages. Opposite states of macrophage activation are classically activated M1 and alternatively activated M2 macrophages, characterized, respectively, by pro- and anti-inflammatory activity. A balance shift towards M2 macrophages has been considered as a new therapeutic strategy of psycho-neurological disorders. One of the inducers of the M2 phenotype is the efferocytosis. We have previously developed an original protocol for the generation of human macrophages under conditions of deficiency of growth / serum factors, in which M2 phenotype is formed through efferocytosis. Macrophages (M2(LS), LS – Low Serum) obtained according to this protocol express M2-associated markers, and are characterized by high production of growth and pro- angiogenic factors (IGF-1, VEGF, BDNF, EGF, FGF-basic, etc.), which can suppress inflammation and stimulate neuroregeneration / neuroplasticity. In the model of stress-induced depression, the antidepressant effect of soluble factors of M2(LS) macrophages was shown, accompanied by a decrease in the level of pro- inflammatory cytokines in certain brain structures. However, the effect of M2(LS) factors on neurogenesis remained unexplored. In the present work, which is a continuation of the aforementioned study, we analyzed the effect of intranasal administration of M2(LS) soluble factors on neuronal density in different brain areas – the frontal cortex and hippocampus – of depression-like mice. The results obtained showed that neuronal density in the frontal cortex, CA1 and CA3 zones of the hippocampus, was significantly higher in mice with intranasal administration of M2(LS) conditioned medium than in depression-like mice, and reached the level of neuronal density in intact animals. These results may indicate the neuroregenerative activity of M2(LS) macrophages in the model of stress-induced depression, which is mediated through soluble factors and manifests itself in an increase in the density of neurons in the brain

M-CSF and GM-CSF determinate fibromodulatory activity of polarized human macrophages

GM-CSF and M-CSF, the hematopoietic colony-stimulating factors, induce various phenotypic changes in macrophage lineage populations and promote cell differentiation, respectively, into M1- and M2-like macrophages. The pro- and anti-inflammatory properties of macrophages generated by these colony-stimulating factors are well described, but the contribution of differentiation and polarization signals to the fibromodulatory activity of macrophages remains unexplored. To clarify the differences in the fibrogenesis regulation mechanisms inherent in differently activated macrophages, we studied the effects of macrophage-conditioned media on proliferation and differentiation of dermal fibroblasts. In this study, the human macrophages generated from peripheral blood monocytes were investigated. They were induced for differentiation by M-CSF or GM-CSF, being further polarized in the M1 direction with lipopolysaccharide and, in the M2 direction, with IL-4 or dexamethasone. Proliferative response of the fibroblasts was determined radiometrically by [3H]-thymidine incorporation. Differentiation into myofibroblasts was determined with flow cytometry technique, as expression of a specific marker α-smooth muscle actin (α-SMA). The level of macrophage TGF-β1 production was assessed using an appropriate ELISA kit. The data obtained indicate that the macrophages differentiated under the influence of “homeostatic” M-CSF are characterized by a moderate stimulating effect upon fibroblast proliferation, and the effects of M2 (IL-4) and M2 (Dex) macrophages exceed that of M1 (LPS), but do not differ significantly from each other. The M-CSF-induced M1 (LPS) and M2 (IL-4) macrophages, but not M2 (Dex), enhance the fibroblast differentiation and show similar level of stimulation. In contrast to M-CSF, the macrophages induced by “pro-inflammatory” GM-CSF exhibit a pronounced stimulatory effect on fibroblast proliferation, and the effects of M2 macrophages exceed those of M1 cells, being most pronounced for M2 (Dex). At the same time, only GM-CSF-induced M2 (IL-4) macrophages enhance fibroblast differentiation. Dexamethasone-polarized macrophages do not significantly affect fibroblast differentiation regardless of the CSF used (M-CSF or GM-CSF). The content of TGF-β1 in the supernatants of differently activated macrophages does not correlate with the level of stimulating effect of macrophage-conditioned media upon fibroblast differentiation. In general, the data obtained suggest the involvement of differentiation and polarization signals into modulation of pro- and anti-fibrogenic properties of macrophages

INTRANASAL INHALATIONS OF BIOACTIVE FACTORS PRODUCED BY M2 MACROPHAGES IN THE TREATMENT OF PATIENTS WITH ORGANIC BRAIN SYNDROME

The aim of present study was to evaluate safety and clinical efficacy of inhalatory immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages applied for treatment of patients with organic brain syndrome (OBS).Materials and methods. The study under the NCT02957123 protocol (www.ClinicalTrails.gov) included thirty patients with OBS of various genesis (10 men and 20 women aged 18 to 81; Me, 62.5 years). Neurological assessment and the levels of 32 cytokines in the blood serum of patients were evaluated before and 2-3 days after completion of inhalation immunotherapy.Intranasal inhalations of cell-free culture medium of M2 macrophages (2 mL, once a day for 28-30 days) were safe and well tolerated. None of 30 treated patients had severe adverse events and serious treatmentrelated side reactions. One month after starting the inhalations, a positive dynamics in neurological status was noted in all the patients. A marked clinical response was documented in twenty out of thirty patients (67%), which manifested as improvement, according to all scales and questionnaires. The neurological improvement was not reversed over 6 months of follow-up period. In other ten patients (33%), a moderate clinical response was shown as improvement of individual scores. The positive changes were as follows: 1) a 43% decrease in anxiety and depression scores (according to HADS scale, pU = 0.0008); 2) an increase of total motor activity (stability and gait) by 25%, pU = 0.0001); 3) correction of cognitive functions (MoCa test, pU = 0.007); 4) reduced number and intensity of the disease symptoms by 52% (pU = 0.0001). This marked clinical response to immunotherapy is shown to be associated with correction/normalization of serum hepatocyte growth factor (HGF) level.Conclusion. Inhalation immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages can improve neurological and functional recovery in patients with organic brain syndrome

IMMUNOMODULATORY EFFECT OF CIRCULATING BONE MARROW PROGENITORS AS A POSSIBLE MECHANISM OF NEUROPROTECTION IN TRAUMATIC BRAIN INJURY

We have previously shown that acute traumatic brain injury (TBI) is accompanied by increased level of circulating bone marrow progenitors, and favorable outcome is associated with early mobilization of CD34+CD45+ hematopoietic progenitor cells (HP). The present study was aimed at investigating whether patients with early HP mobilization differed from those with mobilization failure by systemic inflammatory reaction and immune parameters. The TBI patients were characterized by increased levels of serum C-reactive protein (CRP), IL-1в, IL-6, IL-8, MCP-1, G-CSF and IL-1ra indicative for presence of systemic inflammatory response. Importantly, patients with lacking mobilization of early HPs were shown to have significantly higher serum levels of CRP, MCP-1, MIP-1в, and G-CSF and a lower level of VEGF. In addition, patients with lack of early HP mobilization differed by significantly lower absolute number of lymphocytes, CD3+ T cells, CD4+ T cells, CD16+ NK cells and proliferative response of mononuclear cells to stimulation with ConA as well as by 4-fold higher rate of infectious complications compared with the opposite group. These data suggest that correlation of early mobilization of CD34+CD45+ cells with a favorable outcome in TBI patients may be partially mediated by anti-inflammatory and immunomodulatory effects of circulating bone marrow progenitors

HUMAN BONE MARROW AND ADIPOSE TISSUE DERIVED MESENCHYMAL STROMAL CELL INFLUENCE ON NEUROLOGICAL DEFICIT RECOVERY IN A MODEL OF SEVERE TRAUMATIC BRAIN INJURY IN RATS

In the present study we characterized the effect of transplantation of bone marrow and adipose tissue MSCs in the model of brain injury in rats. This study was performed on Wistar rats (males and females in equal proportions with body weight of 250—270 g; n = 50). Traumatic brain injury was produced, by original spring-loaded, mechanism to precise dosing of impact force. Neurological disorders were assessed by Chen scale, a vertical grid test and a Morris water maze. MSC injections on day 1 were accompanied by a significant reduction in neurological deficit in compare with the control group. Adipose tissue derived MSCs were found to have a more pronounced effectiveness than MSCs obtained from bone marrow

Expression of M2-associated molecules in circulating monocyte subsets in fertile non-pregnant women and pregnant women with uncomplicated pregnancy

In humans circulating monocytes include classical (CD14++CD16- ), intermediate (CD14++CD16+) and non-classical/alternative (CD14+CD16++) monocytes, which in turn can be activated via the classical or alternative pathway. Pregnancy is accompanied by significant changes in the monocyte compartment, which is manifested by an increase in the number of circulating monocytes, including the proportion of intermediate monocytes, and a change in their function. However, the functional properties of monocyte subsets during gestation remain largely unexplored. We hypothesized that circulating monocytes may be activated in an alternative pattern and acquire features of M2 polarization (anti-inflammatory / immunosuppressive properties). The aim of the investigation was to study M2-associated markers that characterize the anti-inflammatory and immunosuppressive potential of myeloid cells in subpopulations of circulating monocytes in fertile nonpregnant women and women with uncomplicated pregnancy in the 2nd trimester. It was shown that in fertile non-pregnant women intermediate and non-classical monocytes are characterized by a higher expression of M2-associated markers (CD206, Arginase 1, MerTK) compared to classical monocytes. In the 2nd trimester of pregnancy, the expression of these molecules on monocytes increases significantly, which is manifested by 1) an increase in the proportion of CD206+ cells in subpopulations of classical and intermediate monocytes, 2) an increase in the mean fluorescence intensity of Arginase 1 in all monocyte subsets, 3) an increase in the proportion of MerTK+ cells in subpopulations of classical and intermediate monocytes and mean fluorescence intensity across all monocyte subsets. The highest content of CD206+ and MerTK+ cells in pregnant women is detected in the subpopulation of intermediate monocytes, and the highest values of the mean fluorescence intensity of Arginase 1 and MerTK – in the subpopulations of intermediate and non-classical monocytes. The data obtained demonstrate that monocytes of pregnant women in the 2nd trimester of pregnancy are characterized by signs of M2 polarization. This is confirmed not only by an increase in the expression of the M2-associated mannose receptor CD206, but also by an increase in the expression of Arginase 1 and MerTK, which mediate the immunosuppressive activity of myeloid cells and, in particular, macrophages of the M2 phenotype. Further studies of M2-associated markers in monocyte subpopulations during gestation will allow a more detailed characterization of the regulatory role of circulating myeloid cells during pregnancy

M2-LIKE MACROPHAGES ARE POTENTIAL CANDIDATES FOR BRAIN STROKE OUTCOMES' TREATMENT

The safety and effectiveness of in vitro generated M2-like macrophages for treatment of patients with ischemic and hemorrhagic brain stroke in reparative and residual periods has been evaluated. A single endolumbar administration of autologous M2-like macrophages in the mean dose of 17,9 х 106 cells was conducted in 13 patients with ischemic (n = 10) and hemorrhagic (n = 3) brain stroke. At 6 months after cells administration all the patients had clinical Improvement. NIHS score decreased from 8,6 ± 1,06 to 4,4 ± 0.55 (p = 0,0008), several patients showed the decrease of sensitiveness impairments, improvement of cognitive functions and enhanced quality of life. Thus, we demonstrated an ability of generation M2-like macrophages from patients with brain stroke. Endolumbar administration of these cells was safe andi didn't cause severe adverse effects and complications andi — in preliminary data — improvedi motional and cognitive functions