NK cells expansion <i>in vitro</i> is followed by loss of inhibitory KIR expression

NK cells are innate lymphocytes that are able to eliminate altered cells, which makes them promising for the immunotherapy of viral diseases and tumors. The NK cell population is characterized by high phenotypic and functional diversity. In particular, in the pool of highly differentiated NK cells in the presence of cytomegalovirus (HCMV), a population of adaptive cells can be formed, characterized by a high lifespan and high cytotoxicity. However, in order to carry out a cytotoxic reaction, a NK cell must undergo a licensing process, during which it acquires the expression of NKG2A and KIRs. Currently, there are many effective methods of NK cell accumulation for subsequent use in therapy, one of them is the stimulation with IL-2 and K562-mbIL21 feeder cells. Highly differentiated adaptive-like NK cells are able to expand in respond to such stimulation. However, the phenotype of actively expanding NK cells dynamically changes. Loss of inhibitory KIR expression during intense proliferation of NK cells may adversely affect their cytotoxic potential. This work shows that highly differentiated CD56dimNKG2C+ NK cells from HCMV-seropositive individuals have a high proportion of KIR2DL2/3+ cells. This may indicate a high stability of KIR receptor expression in this population. We have shown that CD56dimNKG2C+ clonal cultures obtained by stimulation with IL-2 and K562- mbIL21 are characterized by high stability of KIR2DL2/3 expression compared to NKG2C-negative and less differentiated CD56brightNKG2C+. Also, in heterogeneous cultures of adaptive NK cells precursors CD57- CD56dimNKG2C+, a higher expression level of KIR2DL2/3 was observed in comparison with NKG2C-negative cultures of CD57-CD56dimNKG2C-. Thus, the accumulation of NK cells upon stimulation with IL-2 and K562- mbIL2 feeder cells can lead to loss of expression of KIR receptors and a decrease in their functional activity. However, cultures of highly differentiated NK cells of HCMV-seropositive individuals CD56dimNKG2C+, as well as cultures of precursors of adaptive NK cells CD57-CD56dimNKG2C+, are characterized by a greater stability of KIR2DL2/3 expression. As a result, stimulation with IL-2 and K562-mbIL21 feeder cells can be used to accumulate adaptive-like cells and their progenitors with stable inhibitory KIR expression and high cytotoxic potential

Early IgE production is associated with accumulation of CD11b+ classical dendritic cells and CD11b+CD11c- macrophages in subcutaneous adipose tissue

In view of increased incidence of IgE-mediated diseases in Russia and worldwide, the efforts of various research groups are focused on studying the mechanisms that trigger the process of switching B-lymphocytes to IgE synthesis upon human exposure to harmless allergens, including the role of various antigen-presenting cells (APCs) in this process. However, the role of distinct APCs upon long-term penetration of low antigen doses via the tissue barriers, is yet poorly understood, as well as specific features of these events upon entrance of the antigen through the subcutaneous adipose tissue which contains tissueassociated lymphoid clusters (TALC).The aim of this work was to determine the relationship between the local accumulation of various APCs in the subcutaneous adipose tissue and development of early IgE production in a clinically relevant experimental model of allergy with long-term administration of low allergen doses. In this experimental model, specific IgE synthesis is induced, with minimal concomitant IgG production, thus mimicking the situation observed in patients with clinically sound allergies. BALB/c mice were immunized for 4 weeks subcutaneously in the withers area or intraperitoneally with low (100 ng) or high (10 μg) doses of the model allergen (ovalbumin). Blood samples were taken weekly from mice for ELISA testing, to determine the production of specific antibodies. Provocation tests were performed with high dose of the allergen, and adipose tissue samples were taken from the site of antigen injection for flow cytometric assays, in order to evaluate the contents of various APC subpopulations. Specific IgE production was induced mainly by subcutaneous injection of the antigen at low doses (100 ng) into the area of withers. When using this experimental regimen, we observed accumulation of classical CD11b+ cells in adipose tissue at the withers site, but not in the peritoneal adipose tissue, in absence of CD11b- classical, inflammatory or plasmacytoid, dendritic cells. These findings coincided in time with increased production of specific IgE on days +14 to +21. Accumulation of CD11b+CD11c- macrophages and their CD206+ M2 subpopulations at early terms (days +7 and +21) was also observed only after subcutaneous injection of the antigen into the withers area. The high-dose antigen injection (10 μg) which mediated IgG1 production to greater extent than production of IgE, led to earlier accumulation of CD11b+ classical dendritic cells (on day 7th), and to the absence of macrophage accumulation at later stages (day 21th). Thus, the early start of specific IgE production upon low-dose injection of the antigen into the subcutaneous adipose tissue may be associated with its presentation by CD11b+ classical dendritic cells in the presence of CD11b+CD11c- macrophages

DISTRIBUTION OF <i>MICA</i> ALLELES IN THE RUSSIAN POPULATION

Stress factors, infections, tumor transformation of the cells of organism induce the expression of MICA protein, which is a ligand for the NKG2D receptor of NK and T cells. The interaction of the NKG2D receptor on the surface of the cells of the immune system with MICA results in activation of lymphocytes and elimination of the ligand carrier. The MICA gene has a high level of polymorphism. To date, 87 alleles have been described; their products differ in ability to activate cytotoxic lymphocytes, that can affect the progression of a number of diseases, such as cancer, viral infections, autoimmune diseases. The distribution of MICA alleles in different ethnic groups varies considerably. The analysis of MICA polymorphism in a current ethnos is necessary for revealing the relationships between certain MICA alleles and different diseases. Goal. This work is aimed at studying of the distribution of MICA alleles in Russian population. Materials and methods. Polymorphism of MICA was analyzed according to the procedure proposed by Yizhou Zoe and Peter Stastny. The procedure included: 1) isolation of genomic DNA from whole blood; 2) PCR for amplification of a fragment of the MICA gene; 3) sequencing of the resulting PCR fragments. Analysis of the results of sequencing was carried out using the programs Vector NTI and Chromas Lite. Results. The genotype of the MICA alleles of 119 donors has been determined. Of the 87 MICA alleles described in the literature, 15 were found among the samples studied. The frequencies of MICA alleles were the following: *002 – 19.3%, *004 – 6.7%, *007 – 3.0%, *008 – 35.7%, *009 – 10.1%, *010 – 5.0%, *011 – 3.8%, *012 – 2.1%, *016 – 2.5%, *017 – 3.4% *018 – 5.5%, *019 – 0.4%, *027 – 1.3%, *053 – 0.8%, *068 – 0.4%. The distribution of MICA alleles in Russia was found to be similar to that of European countries. When comparing literary data for different countries of the world, it was found that the differences in the distribution of MICA alleles are expressed mainly between races, and not nations. Conclusions. In this paper, the distribution of MICA alleles in Russian population has been analyzed. It turned out to be very similar to those of other European countries and has a number of significant differences from the ethnoses of the Mongoloid race (Japan, China, Korea). The analysis of the distribution of MICA alleles in the Russian population may be useful for identifying the predisposition of individuals to certain diseases

Сочетанная анестезия при коронарном шунтировании без искусственного кровообращения у пациентов с висцеральным ожирением

The objective: to analyze results of the use of combined anesthesia for coronary artery bypass grafting without cardiopulmonary bypass (off-pump CABG) in patients with visceral obesity (VO). Subjects and methods. A randomized study of results of surgical treatment in patients with VO and coronary heart disease who underwent off-pump CABG was conducted. 197 patients were included in the study. The main group (n = 98) included patients who underwent combined anesthesia (with thoracic epidural analgesia (TEA)) and patients in the control group (n = 99) underwent total intravenous anesthesia (with narcotic analgesics). Results. In patients from the main group, a significant decrease in the incidence of acute kidney injury (p = 0.0180), respiratory complications (p = 0.0177), atrial and ventricular arrhythmias (p = 0.0029) was recorded. With the use of TEA, the duration of treatment of patients in the intensive care unit (p = 0.0229) and duration of hospital stay (p = 0.0419) significantly decreased. Conclusion: The use of combined anesthesia (with TEA) for off-pump CABG in patients with visceral obesity reduces the risk of early postoperative complications, the duration of hospital stay and treatment in the intensive care unit.Цель: провести анализ результатов применения сочетанной анестезии при коронарном шунтировании без искусственного кровообращения (off-pump КШ) у пациентов с висцеральным ожирением (ВО). Материал и методы. Проведено рандомизированное исследование результатов хирургического лечения больных ишемической болезнью сердца с ВО, которым выполнено off-pump КШ. В исследование включено 197 пациентов. Основную группу (n = 98) составили пациенты, у которых выполняли сочетанную анестезию (с грудной эпидуральной анальгезией ‒ ГЭА), больным контрольной группы (n = 99) проводили тотальную внутривенную анестезию (с наркотическими анальгетиками). Результаты. У пациентов основной группы зафиксировано статистически значимое снижение частоты развития острого повреждения почек (p = 0,0180), респираторных осложнений (p = 0,0177), предсердных и желудочковых аритмий (p = 0,0029). На фоне применения ГЭА статистически значимо снизилась длительность лечения больных в палате интенсивной терапии (p = 0,0229) и послеоперационной госпитализации (p = 0,0419). Вывод. Применение сочетанной анестезии (с ГЭА) при off-pump КШ у пациентов с ВО позволяет снизить риск развития ранних послеоперационных осложнений, длительность послеоперационной госпитализации и продолжительность лечения в палате интенсивной терапии

Umbilical cord blood as a promising source of NK cells for immunotherapy

Currently, a large number of studies on genetic modification of cord blood NK cells (UCB-NK) are carried out at both clinical and preclinical levels. Immunotherapy based on UCB-NK cells has great potential for antitumor therapy. However, despite having known several advantages over peripheral blood NK cells (PB- NK), including a high concentration in cord blood and low virulence rate, UCB-NK cells are predominantly characterized in the scientific literature as immature and low-functioning NK cells. In this work, we studied the phenotypic characteristics of UCB-NK cells and the possibility of stimulatory compensation of the decreased functional activity of UCB-NK cells. Our studies revealed UCB-NK cells can be characterized as poorly differentiated and weakly activated cells with high level of inhibitory receptor NKG2A and low level of activating receptor NKG2C and HLA-DR, accordingly with the literature data. Two types of stimuli were chosen to stimulate freshly isolated UCB-NK cells: 1) 100 units of IL-2; 2) combinations of 100 units IL-2 and K-562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). It was shown the degranulation (LAMP-1) and proliferative activity was higher than for parallel cultured ex vivo PB-NK cells under the same conditions for UCB-NK cells stimulated for 7 days with IL-2 + K562-mbIL21. Moreover, stimulation in the way of IL-2 + K562-mbIL21 seemed to be a more perspective way to obtain a large number of proliferatively active UCB-NK cells compared to stimulation with IL-2 only. Since genetic modification of NK cells is a promising way to improve the antitumor properties of NK cells, retroviral transduction procedure was performed to study of the stimulated UCB-NK cells. UCB-NK cells stimulated with IL-2 + K562-mbIL21 were transduced on day 8 of cultivation. In this study, we used targeted overexpression of the adaptor molecule DAP12, which is involved in the signaling of activating NK cell receptors. PB-NK cells and UCB-NK cells were transduced under the equal experimental conditions in same volume of viral particles. As a result, the transduction efficiency was found to be more than 4-fold higher for UCB-NK cells compared to PB-NK cells. Thus, UCB-NK cells appear to be a promising tool for further research in cancer immunotherapy

Unconfined Aquifer Flow Theory - from Dupuit to present

Analytic and semi-analytic solution are often used by researchers and practicioners to estimate aquifer parameters from unconfined aquifer pumping tests. The non-linearities associated with unconfined (i.e., water table) aquifer tests makes their analysis more complex than confined tests. Although analytical solutions for unconfined flow began in the mid-1800s with Dupuit, Thiem was possibly the first to use them to estimate aquifer parameters from pumping tests in the early 1900s. In the 1950s, Boulton developed the first transient well test solution specialized to unconfined flow. By the 1970s Neuman had developed solutions considering both primary transient storage mechanisms (confined storage and delayed yield) without non-physical fitting parameters. In the last decade, research into developing unconfined aquifer test solutions has mostly focused on explicitly coupling the aquifer with the linearized vadose zone. Despite the many advanced solution methods available, there still exists a need for realism to accurately simulate real-world aquifer tests

Интегральный способ прогнозирования риска панкреатита после эндоскопической ретроградной холангиопанкреатографии

The objective: development of a method for determining the individual risk of post-ERCP pancreatitis (PEP) before endoscopic intervention.Subjects and Methods. A prospective observational study of the results of therapeutic endoscopic retrograde cholangiopancreatography (ERCP) was performed in 1,210 patients. The primary end point was the development of PEP. Multiple regression logistic analysis was used to determine the risk of developing PEP.Results. A model of independent variables was determined, which significantly (p = 0.0001) affected the risk of developing PEP. Based on the results of the logistic analysis, regression coefficients were calculated for all significant patient-dependent factors (gender, age, preoperative level of amylasemia and bilirubinemia, and nature of the underlying disease). The risk of PEP decreased in men (by 47.5%), with an increase in the age of patients (for 1 year by 2.3%) and bilirubin (with an increase of 1 μmol/l by 0.3%) but increased with an increase in amylase activity blood serum (by 0.7% for each 1 U/l). A formula has been developed to assess the individual risk of PEP at the stage of preparing a patient for therapeutic ERCP.Conclusion. Analysis of patient-dependent factors makes it possible to reliably determine the risk of developing PEP. It was determined that a female gender, young age (under 40 years old), sphincter of Oddi dysfunction increased the risk of PEP, while a male sex, stenting of the main pancreatic duct, and jaundice reduced this risk. The use of the proposed formula will help predict a significant (p = 0.0001) degree of risk of PEP before performing primary therapeutic ERCP in a particular patient.Цель: разработка способа определения индивидуального риска развития пост-ЭРХПГ-панкреатита (ПЭП) до выполнения эндоскопического вмешательства.Материал и методы. Проведено проспективное наблюдательное исследование результатов лечебной эндоскопической ретроградной холан- гиопанкреатографии (ЭРХПГ) у 1 210 пациентов. Первичной конечной точкой считали развитие ПЭП. Для определения риска развития ПЭП применяли множественный логистический регрессионный анализ.Результаты. Определена модель независимых переменных, которая достоверно (p = 0,0001) влияет на риск развития ПЭП. По результатам логистического анализа вычислены регрессионные коэффициенты для всех значимых пациент-зависимых факторов (пол, возраст, предоперационный уровень амилаземии и билирубинемии, характер основного заболевания). Риск ПЭП снижался у мужчин (на 47,5%), при увеличении возраста пациентов (за 1 год на 2,3%) и уровня билирубина (при увеличении на 1 мкмоль/л на 0,3%), но повышался при увеличении активности амилазы сыворотки крови (на 0,7% на каждую 1 Ед/л). Разработана формула, позволяющая оценить индивидуальный риск ПЭП на этапе подготовки пациента к выполнению лечебной ЭРХПГ.Вывод. Анализ пациент-зависимых факторов позволяет достоверно определить риск развития ПЭП. Определено, что женский пол, молодой возраст (до 40 лет), дисфункция сфинктера Одди увеличивали, а мужской пол, стентирование главного панкреатического протока, наличие желтухи снижали риск развития ПЭП. Использование предложенной формулы поможет прогнозировать достоверную (p = 0,0001) степень риска ПЭП до выполнения первичной лечебной ЭРХПГ у конкретного пациента

NOVOSIBIRSK REGION PROGRAM OF THE LIVER TRANSPLANTATION IN CHILDREN

The aim of the study was to analyze the early and late outcomes of pediatric liver transplantation (LTx) program in the Novosibirsk region. Material and Methods. The retrospective study included 24 patients aged 72 ± 74.8 months (from 4 to 212 months, median – 20 months) with a body weight of 21.7 ± 18.1 kg (from 4.5 to 55 kg, median – 12.5 kg). Two (8.3 %) cadaveric whole liver grafts, 19 (79.2 %) living donor liver grafts, and 3 (12.5 %) cadaveric liver fragments (reduced-size or split-liver) were transplanted. Results. The features of vascular and biliary reconstruction in different types of LTx are discussed. The incidence of vascular and biliary complications was 8.3 and 20.8%, respectively. The patients stayed in the intensive care unit for 9 ± 5.1 days (from 4 to 22 days, median 8 days) and total length of hospital stay was for 40 ± 25.4 days (from 19 to 136 days, median 32 days). Two (8.3 %) recipients had early graft dysfunction. Perioperative mortality (up to 90 days) was absent. The overall 5-year patient and graft survival rates were 95 and 88 %, respectively. Conclusion. The Novosibirsk region has a pediatric LTx program with outcomes comparable to the data of the leading world and Russian centers

Early maternal mirroring predicts infant motor system activation during facial expression observation.

Processing facial expressions is an essential component of social interaction, especially for preverbal infants. In human adults and monkeys, this process involves the motor system, with a neural matching mechanism believed to couple self- and other-generated facial gestures. Here, we used electroencephalography to demonstrate recruitment of the human motor system during observation and execution of facial expressions in nine-month-old infants, implicating this system in facial expression processing from a very young age. Notably, examination of early video-recorded mother-infant interactions supported the common, but as yet untested, hypothesis that maternal mirroring of infant facial gestures is central to the development of a neural matching mechanism for these gestures. Specifically, the extent to which mothers mirrored infant facial expressions at two months postpartum predicted infant motor system activity during observation of the same expressions at nine months. This suggests that maternal mirroring strengthens mappings between visual and motor representations of facial gestures, which increases infant neural sensitivity to particularly relevant cues in the early social environment