Pressure ulcers represent most common surgical pathology in the pattern of nosocomial complications. However, no unified mechanism leading to development of pressure ulcers has been proposed yet despite the lead role played by compression and immobilization. In this regard, examining immune system as the main component maintaining integrity of anatomical barriers in the skin and mucous membranes seems highly promising for creating new methods to prevent and treat pressure ulcers. Our study was aimed at investigating phenotypic profile of peripheral blood B cells in patients with pressure ulcers. There were enrolled 67 patients complicated with pressure ulcers at the Department of Surgery. Examination of pressure ulcers included determining anatomical localization, investigating depth (degree) and size of the lesion (by measuring wound area), skin color and assessing nature of pressure ulcer edges and edema, wound bottom, presence of cavity with tendons and/or bone formations may be recognized therein, characteristics of exudate (smell, color), pain sensation. In control group there were included 81 apparently healthy subjects. All groups contained age- and sex-matched subjects. Phenotyping of peripheral blood B cells was performed by using flow cytometry with panel of monoclonal antibodies. It was found that count of B cells in patients did not change in parallel with increased total lymphocyte count, but was associated with their functional activity (increased percentage of CD23- and CD38-positive B cells). Moreover, percentage of B1 and naive B2 cells declined in patients with pressure ulcers that seemed to be associated with the premorbid background of the main disease, lack of adequate wound healing process coupled to tissue necrosis and damage to skin capillaries. The more severe the clinical course of pressure ulcers (regarding area and stage of development), the smaller percentage of B2 cells (both naive and memory B cell subsets) was detected in the patient peripheral blood. At the same time, changes in the B cell phenotypic profile from patients are associated with the area of lesion, but not with the stage of developing pressure ulcers evidencing that B cells affect healing of pressure ulcers. The features of B cell phenotype promote unfavorable disease outcome evidenced by the lack of quantitative differences in B cell lineage composition or level of surface expression for activation markers.

Цель. Исследовать уровень аллоиммунизации к антигенам систем HNA у доноров крови и её компонентов. Методы. В исследование были включены образцы крови 1127 доноров, дающих кровь и её компоненты в ФГБУ РосНИИГТ ФМБА России: 635 мужчин и 492 женщин. Доноров-женщин в возрасте 18–30 лет было 36,18% (n=178), в возрасте 30–60 лет — 63,82% (n=314). Все обследованные доноры не имели в анамнезе трансфузий компонентов крови. Скрининг и идентификацию аллоантител к антигенам HNA проводили в непрямом тесте иммунофлюоресценции гранулоцитов методом проточной цитофлуориметрии с использованием моноклональных антител CD16-PE, CD45-PC7, CD19-APC, Goat F(ab')2 Anti-Human IgG-FITС и панели донорских нейтрофилов с установленным HNA-генотипом. Результаты. Частота аллоантител к антигенам систем HNA у обследованных доноров составила 0,35% (n=4). В группе доноров-мужчин аллоантитела к HNA выявлены не были. Все сенсибилизированные доноры были женского пола в возрасте 30–60 лет. Частота антител к HNA у доноров-женщин составила 0,81%. Выявленные антитела имели специфичность анти-HNA-1а, анти-HNA-3b и анти-HNA-4b и принадлежали к иммуноглобулинам класса G. У одного из сенсибилизированных доноров специфичность антител установить не удалось. Для выявления аллогенной природы антител у иммунизированных доноров были определены генотипы HNA. Вывод. Аллоантитела к антигенам систем HNA встречаются редко и определяются у 0,35% доноров крови и её компонентов; аллоантитела были выявлены только у доноров-женщин с частотой 0,81% и направлены против HNA-1а, HNA-3b и HNA-4b; генотипирование HNA позволило подтвердить аллогенную природу антител.

At present, the role of intestinal microbiota in diverse diseases of the central nervous system, including of multiple sclerosis (MS) has been extensively investigated. Self-reactive CD4+ Th1 and Th17 cells specific to myelin-derived antigens play a key role in the MS pathogenesis. Taking into consideration pathogenetic features related to MS development, we examined a relation between intestinal microbiocenosis and abundance of various peripheral blood helper T (Th) cell subsets in MS patients. Objective of the study: to assess prevalence of individual members of the intestinal microbiota in MS patients and analyze a relation with peripheral blood Th cell subsets. Prevalence of symbiotic and opportunistic microbial species was estimated by bacteriological method and real time PCR in 112 MS patients (72 females, 40 males) of varying severity and duration. Th cell subsets (Th1, Th2, Th17, Th1/Th17, Th17/Th22, DP Th17) were analyzed by using multi-color flow cytometry based on Th cell subset-specific surface expression of chemokine receptors. A relationship between individual intestinal microbiota species and severity, duration and rate of MS progression, as well as with the phenotype of immune cells was assessed. It was found that the most significant correlation between percentage of peripheral blood Th cell subsets was observed with prevalence of Lactobacillus spp., Enterococcus spp. and Enterobacter spp. Moreover, prevalence of Enterococcus spp. Th cell composition influenced synergistically or antagonistically together with Enterobacter spp. or Lactobacillus spp., respectively. It is suggested that direct and indirect impact of intestinal microbiota composition on human immune system might contribute to developing novel strategies for treating MS.

The aim of the study was to investigate an interdependence between the phenotype of dendritic cells (DC) differentiated from monocytes and the number of pro-inflammatory monocytes in peripheral blood of patients with kidney cancer (KC). The study involved 28 patients at the age of 40-55 years suffering with KC (Т3N0М0, clear cell type) before surgical treatment. The diagnosis was verified histologically. 31 healthy agematched persons were examined as a control group. Mononuclear cells were isolated from heparinized venous blood by centrifugation in a Histopaque®-1077 density gradient followed by plastic adsorption in RPMI 1640 medium supplied with 10% autologous serum. Immature DCs (iDCs) were generated from blood monocytes by culturing for 5 days with GM-CSF and IFNα. Activation of DCs (mDCs) was induced by incubation with the tumor cell lysate and TNFα, followed by incubation for 48 hours. A tumor fragment was used to prepare the lysate of autologous tumor cells. Phenotyping of blood monocytes and DC at various maturation stages was performed by flow cytometry. The numbers of CD14+CD16+ monocytes in peripheral blood of KC patients were decreased (up to 42% of the total monocyte level) against the control ranges. In this regard, the analysis of the dependence between the phenotype of DCs differentiated from monocytes and the number of pro-inflammatory blood monocytes was carried out by comparing the groups with a high content of pro-inflammatory monocytes in the blood in KC patients (> 42%, near-control range) and low content (resp., 42%) of proinflammatory monocytes in blood. The phenotype of dendritic cells in KC patients with different content of proinflammatory monocytes during maturation/activation showed more differences. In the patients with low levels of pro-inflammatory monocytes, the cell pool of in vitro maturing DCs was characterized by low level of CD86 and HLA-DR receptor expression, thus reflecting a weak co-stimulating and antigen-presenting activity. In the patients with high levels of pro-inflammatory monocytes in blood, the in vitro activated DCs showed higher level of functional activity using the above markers. The revealed differences in the DC phenotype and interrelations with amounts of blood monocyte subpopulations in KC patients may presume the programmed cell differentiation mechanisms depending on the microenvironment, under pathogenic conditions (i.e., in presence of malignant tumor growth).

Tuberculosis (TB) is one of the most common infections worldwide. Eradication of an intracellular pathogen M. tuberculosis requires to induce a Th1 response by activating IFNγ-producing tissue macrophages. Along with Th1 cells, various subsets of Th17 and follicular T-helper cells (Tfh) able to secrete a broad range of cytokines, including IFNγ, can also be involved in eliminating bacterial pathogens. It justified analyzing in this study changes in percentage of various peripheral blood Th subsets, including Th1, Th2, Th17 and Tfh cells, in TB patients. For this, major CD3 + CD4 + T cell subsets were assessed by using multicolor flow cytometry in TB patients (n = 40) and healthy volunteers (n = 30). It was found that in TB patients vs. control group percentage of peripheral blood CD45RA – CCR7+ central memory (CM) Th was decreased also affecting frequency of some functional T cell subsets, e.g. either lowering Th2 cells (9.11% (6.95; 13.77) vs. 7.21% (5.64; 9.84), p = 0.012) or elevating CCR6+ Th17 subsets (35.92% (27.72; 41.06) vs. 40.39% (35.41; 47.79; p = 0.016), respectively, but not influencing Th1 and Tfh subsets frequencies. Moreover, percentage of total CCR6 + CM Th cells in TB patients vs. control was decreased in CCR4 – CXCR3 + Th17.1 cell subset (42.87% (33.64; 49.45) vs. 52.26% (46.45; 56.95), p < 0.001), whereas standard CCR4 + CXCR3 – Th17 and CCR6+ DP Th17 subsets were elevated (p = 0.005 and p = 0.002, respectively). In addition, altered Tfh subset composition associated with the increased (p = 0.021) percentage of CXCR3–CCR6 – Tfh2 cells, but decreased CXCR3 + CCR6 – Tfh1 cells (p = 0.036) was observed. Finally, frequency of peripheral blood Th subsets noted above was also analyzed within effector memory (CD45RA – CCR7 – ) cells. It was found that in TB patients vs. volunteers frequency of Th17.1 cells was also significantly lower (p = 0.006) in CCR6 + EM Th (54.43% (41.19; 91.92) vs. 61.76% (54.01; 65.63), whereas percentage of double-positive Th17 was significantly increased (20.83% (15.12; 30.87) and 12.93 % (9.80; 19.01), respectively, p < 0.001). Thus, it suggests that during M. tuberculosis infection percentage of IFNγ-producing Th17 and Tfh cells was reduced compared to control group also affecting both central memory Th cells patrolling peripheral lymphoid organs as well as effector memory Th cells able to exit to site of infection.

Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system. Recent reports have shown CD3 + CD8 + and their different subsets take part in neuroinflammation. During the current study frequency, phenotype as well as CD56 and CD57 expression of peripheral blood CD3 + CD8 + cells derived from healthy volunteers (n = 52), MS subjects during relapse (n = 31) or remission (n = 20) were characterized using ten-color flow cytometry. CD3 + CD8 + were divided into naive, central (CM) and effector memory (EM), as well as effector cells (TEMRA) based on the expression of CD45RA, CD62L, CD27 and CD28. No differences were observed during the comparison of N, CM and TEMRA CD3 + CD8 + subsets relative and absolute number between the groups, but the number of CM cells was decreased in MS groups The relative and absolute number of “naive” CD3 + CD8 + cells were inversely related (r = –0,512, р = 0,003 and r = –0,430, р = 0,014, respectively) to the degree of disability of examined patients. The percentages of CD57 + cells were increased only within naive and CM subsets in both MS groups compared with healthy volunteers. While the relative numbers of CD56 + cells were significantly evaluated in all investigated cytotoxic T cell subsets in MS group with the exception of TEMRA cells from patients during remission in comparison with control group. Our data demonstrate that CD56 level on CD3 + CD8 + subsets may represent a biomarker of neuroinflammation in MS patients.

Tuberculosis is the leading cause of death among HIV infected individuals. In this regard, an important task is the timely detection of tuberculosis in HIV infected patients. Previously, we have shown that the diagnostic value of in vitro test, QuantiFERON-TB Gold In-Tube is not decreased in patients with HIV infection against the background of tuberculosis. However, it remains unclear what kind of cell populations produce IFNγ in response to specific Mycobacterium tuberculosis antigens stimulation in vitro, because the immunodeficiency, caused by HIV, makes primarily for a decrease the abundance and attenuation functions of CD4 T-lymphocytes. The aim of thшы work was to compare the degree of differentiation of T-lymphocytes CD4 (Th) and CD8 (Tcyt) in patients with pulmonary tuberculosis and healthy donors against the background of HIV infection. The study data were obtained during the examination of 28 patients with pulmonary tuberculosis without HIV infection (HIV–TB+), 23 patients with HIV infection (TB–HIV+) and 30 patients coinfected with HIV and tuberculosis (TB+HIV+). The comparison group consisted of 37 healthy individuals (TB–HIV–). Аbsolute and abundance (relative content) of major subpopulations of T-lymphocytes (based on the expression of CD27 marker, CD28, CD45RA and CD62L) in the peripheral blood for all patients included in the study (n = 118) were evaluated by flow cytometry approach. For patients with pulmonary tuberculosis (n = 58) QuantiFERON-TB Gold In Tube (Qiagen, QFT) test was performed. Th/Tcyt ratio was not significantly different among the groups of TB–HIV– and TB+HIV– (1.76 [1.51; 2.30] against 1.86 [1.22; 2.79], p = 0.960). At that time, the size of both subpopulations “terminally differentiated” Tcyt (Tcyt Eff, CD27–CD28– CD62L–CD45RA–) Th lymphocytes and effector memory lymphocytes (Th EM, CD27–CD28+CD62L–CD45RA–), was significantly different in all four study groups. Multidirectional changes of the absolute and abundance (relative content) in these cell populations in comparison with healthy donors for tuberculosis and HIV infection was noticed. Absolute content of Tcyt Eff, compared with healthy donors (76.1 [20.7; 143.5]), 4-fold increases in the group of HIV+TB+ and 2 times in groups TB+HIV– and TB–HIV+. Th EM content increases only at TB+HIV– group compare to healthy donors. In groups of patients with HIV infection (TB–HIV+ and TB+HIV+) a decrease in the content of these cells was observed. Thus, our work shows that the population of Th EM and Tcyt Eff could potentially be viewed as universal biomarkers for two socially significant infectious diseases: tuberculosis and HIV infection. In future experiments, it is necessary to validate these results to ensure specificity and determine the number of Th EM and Tcyt Eff specificly induced by Mtb antigens.

Цель. Оценка влияния комплекса пробиотических лактобактерий на эффективность лечения и нормализацию иммунных нарушений при папилломавирусной инфекции у женщин. Методы. Обследованы 65 пациенток репродуктивного возраста с патологией шейки матки и папилломавирусной инфекцией. В качестве источника комплекса пробиотических лактобактерий использовали препарат «Проваг». Иммунологические исследования были проведены до и после лечения. Исследование фенотипа лимфоцитов крови проводили методом проточной цитометрии. Концентрацию иммуноглобулинов А, М и G в сыворотке крови определяли иммуноферментным методом. Результаты. У больных папилломавирусной инфекцией в 1-е сутки обследования обнаружено снижение процентного количества Т- и NK-клеток. Независимо от метода лечения к 30-м суткам наблюдения количество T-лимфоцитов в крови у женщин с папилломавирусной инфекцией повышается. Процентное содержание NK-клеток при традиционной схеме лечения папилломавирусной инфекции к 30-м суткам наблюдения остаётся пониженным. При дополнительном применении комплекса пробиотических лактобактерий у больных папилломавирусной инфекцией к концу лечения выявляется повышение количества NK-лимфоцитов. В 1-е сутки лечения происходит снижение процентного и абсолютного содержания T-лимфоцитов-хелперов. Независимо от метода лечения к 30-м суткам наблюдения количество Т-хелперов увеличивается до контрольного уровня. Уровни концентраций иммуноглобулинов M и G в крови женщин с папилломавирусной инфекцией повышены уже в 1-е сутки лечения. К концу периода наблюдения их концентрации у больных с традиционной схемой лечения остаются повышенными. При дополнительном назначении препарата «Проваг» концентрация данных классов иммуноглобулинов снижается до контрольного уровня. Выводы. В результате проведённого лечения произошло восстановление уровня NK-клеток и Т-хелперов при сохранении повышенной концентрации иммуноглобулинов M и G.