ROLE OF CYTOXIC T-LYMPHOCYTES IN THE PATHOGENESIS OF PRETERM BIRTH

Currently, the existence of a wide range of subpopulations of CD8+T-lymphocytes has been revealed, among which there are subpopulations of naive and effector cells, as well as memory cells. CD8+T-lymphocytes are thought to be a population of lymphocytes with high cytotoxic activity, which is of extreme importance during pregnancy. Given that each subpopulation is characterized by a set of produced mediators, surface and intracellular markers, we can assume their role in the pathogenesis of preterm birth. This determined the need to investigate the role of naive cells, effector cells, and memory cells in the development of spontaneous preterm birth. Data on the content of naive CD8+-lymphocytes in the peripheral blood of women with threatened preterm birth are practically absent. It was found that the infiltration of CD8+-lymphocytes in the area of uteroplacental contact was associated with the development of timely delivery. Chronic chorioamnionitis is the most common condition in idiopathic preterm birth and is characterized by the infiltration of maternal CD8+Tcells into the chorioamniotic membranes. Currently, it is believed that chronic inflammatory lesions of the placenta represent maternal antifetal rejection. This led to the study of the role of these cells in the pathogenesis of preterm birth. Purpose. To establish a possible pathogenetic mechanism of preterm birth in women with threatened preterm birth on the basis of the revealed features of differentiation and functional activity of CD8+- lymphocytes at the systemic levelMaterials and methods. The survey of women was carried out on the basis of the Federal State Budgetary Institution “V. Gorodkov Ivanovo Research Institute of Maternity and Childhood” of the Ministry of Health of the Russian Federation. A total of 126 women were examined, which were retrospectively divided into 2 main groups – women with threatened preterm birth(n = 68), which was divided into 2 subgroups – with the outcome of pregnancy preterm birth (n = 30) and timely delivery (n = 38). The control group included 58 women with uncomplicated pregnancy and who gave birth on time. In the CD8+-lymphocyte population, the content of central – Tcm (CD45RACD62L+), preterminally differentiated-Tem (CD45RACD62L- ) and terminally differentiated-Temra (CD45RA+CD62L- ) memory cells was determined. In all memory cell populations, the content of cells producing Granzyme B intracellularly was determined. The studies were performed using monoclonal antibodies (mAT) by flow cytometry on a FACSCanto II cytometer using the FACSDiva software (Becton Dickinson, USA).The analysis of the features of the relative content of CD8+-lymphocytes in the main group of women, depending on the outcome of pregnancy, was carried out. When comparing patients with a clinic of threatened preterm birth, whose pregnancy ended prematurely, a higher content of CD8+-lymphocytes was revealed than in group c of women who gave birth in a timely manner, which indicates a high stimulation of cytotoxic T-lymphocytes in this group of women. With threatening preterm birth, there is an increase in the content of naive CD8+-lymphocytes in the peripheral blood. Data on the content of naive CD8+-lymphocytes in the peripheral blood of women with threatened preterm birth are practically absent. The increase in CD8+Tn levels is more pronounced in the subgroup of women with a favorable pregnancy outcome. Given this fact, it can be assumed that in women with preterm birth, a lower CD8+Tn is associated with their increased differentiation into effector T-lymphocytes with their subsequent migration to the placental zone. This process could determine the observed decrease in the level of terminally differentiated granzyme-producing CD8+-lymphocytes in a subgroup of women with a pregnancy outcome of preterm birth, which coincided with the literature data

BASOPHILE ACTIVATION TEST FOR THE DIAGNOSTICS OF FUNGAL SENSITIZATION IN THE PATIENTS WITH CYSTIC FIBROSIS

Aspergillus fumigatus colonization in the patients with cystic fibrosis (CF) may cause sensitization against A. fumigatus and/or allergic bronchopulmonary aspergillosis (ABPA), which significantly worsens the course of underlying disease. At the present time, new diagnostic tests are searched for detection of fungal sensitization in these patients. The aim of this work was to evaluate an opportunity of application of basophile activation test with A. fumigatus allergen in vitro using flow cytometry, aiming for identification of fungal sensitization in the CF patients. The study included 190 patients with CF aged 1 to 37 years. All the patients underwent common allergy screening (skin tests with fungal allergens, determination of serum levels of total IgE and specific IgE for the fungal allergens), and mycological examination (microscopy and culture of respiratory substrates). Computed tomography of the chest was performed upon clinical indications. The basophil activation test with the A. fumigatus allergen was performed in 10 CF patients with ABPA, and 10 CF patients without ABPA, in addition to the standard allergological examination. Frequency of sensitization to A. fumigatus in the patients with cystic fibrosis was 27%, the incidence of allergic bronchopulmonary aspergillosis was 5.7%. The number of eosinophils, total IgE and specific IgE levels in CF patients with ABPA were significantly higher than in CF patients without ABPA. In blood of the ABPA patients we have identified 68.5 (52.5-81.5%) of basophilic leukocytes activated by A. fumigatus allergen, with a stimulation index of 17.07 (10.30-27.70). In appropriate comparison group, the stimulation index did not exceed 1.5 (p = 0.000). Direct positive correlation between the levels of specific IgE to A. fumigatus and the number of basophils activated by A. fumigatus allergens was revealed (r = 0.77; р < 0.05). FVC values and the body mass index in CF patients with ABPA were significantly lower when compared with the patients without fungal sensitization. Introduction of the basophil activation test, along with standard techniques, may enable a more differentiated assessment of ABPA development in CF patients. Timely detection of associations between A. fumigatus sensitization and clinical status of CF patients will facilitate early and effective administration of specific therapy

Тяжелый грипп как фактор риска развития инвазивного аспергиллёза лёгких (клинический случай)

During  last  years  the  frequency  of  invasive  pulmonary aspergillosis  (IPA)  in  immunocompetent  patients  has  increased. Clinical case report of successful treatment invasive aspergillosis  with  influenza  A(H1N1)  presented  in  the  article. We analyzed the special literature of patients with IPA following influenza infection. The timely identification and treatment of these patients are necessary.В последние годы увеличилась частота инвазивного аспергиллеза у больных без типичных факторов риска. В статье представлен случай успешного лечения инвазивного аспергиллёза лёгких, возникшего на фоне гриппа A(H1N1), и обзор литературы. Показана необходимость своевременной диагностики и адекватной антимикотической терапии у больных тяжелым гриппом

АСПЕРГИЛЛЕЗ ЛЕГКИХ У БОЛЬНЫХ МУКОВИСЦИДОЗОМ В РОССИЙСКОЙ ФЕДЕРАЦИИ

The aim: to assess the incidence of various forms of pulmonary aspergillosis in patients with cystic fibrosis.Materials and methods. In 2014-2017 yy. in prospective study in  different regions of Russia were included 190 patients with cystic  fibrosis aged 1 to 37 years. Children – 130, adults – 60. All patients  underwent allergy (skin tests with fungal allergens, total IgE level,  specific IgE to fungal allergens) and mycology (microscopy and  cultural investigations of respiratory biomaterials) testing. Chest  computed tomography was performed according to the indications.Results. The incidence of fungal sensitization in patients with cystic  fibrosis was 57%, to Aspergillus spp. – 27%. The incidence of  allergic bronchopulmonary aspergillosis was 5,7%, chronic lung  aspergillosis – 4,2%, invasive aspergillosis developed in one patient  (0,5%) during immunosuppressive therapy after liver transplantation.Conclusion. The incidence of pulmonary aspergillosis in patients  with cystic fibrosis in the Russian Federation was first determined  (10.5%). Mycology testing is indicated for patients with cystic  fibrosis for early treatment of different variants of pulmonary aspergillosis.Цель. Оценить частоту развития различных форм аспергиллеза легких у больных муковисцидозом.Материалы и методы. В 2014–2017 гг. в проспективное исследование в разных регионах  РФ включили 190 больных муковисцидозом в возрасте от 1 до 37 лет (130 детей, 60  взрослых). Всем пациентам провели аллергологическое обследование (кожные тесты с  грибковыми аллергенами, определение уровня общего IgE и специфических IgE к  грибковым аллергенам) и микологическое обселедование (микроскопия и посев  респираторных субстратов). По показаниям выполняли компьютерную томографию органов грудной клетки.Результаты. У больных муковисцидозом частота микогенной сенсибилизации составила  57%, к Aspergillus spp. – 27%. Аллергический бронхолегочный аспергиллез выявили у 5,7%  пациентов, хронический аспергиллез легких – у 4,2%, инвазивный аспергиллез развился у  одного пациента (0,5%) на фоне иммунодепрессивной терапии после трансплантации печени.Заключение. Впервые определена частота развития аспергиллеза легких у больных  муковисцидозом в Российской Федерации (10,5%). Больным муковисцидозом показано  микологическое обследование для своевременного лечения различных вариантов аспергиллеза легких

Инвазивный аспергиллез у взрослых пациентов с ревматическими заболеваниями

Objective: to study risk factors for invasive aspergillosis (IA), its etiology, clinical manifestations, and treatment efficiency in patients with rheumatic diseases (RD).Patients and methods. The first study of proven and probable IA (EORT/MSGERC, 2019) was conducted in 18 patients with RD, who accounted for 3% of all adult IA patients (n=699) included in the 1998–2020 registry of the Department of Clinical Mycology, Allergology, and Immunology, I.I. Mechnikov North-Western State Medical University (Group 1). This group comprised 56% women; the median age was 59 [21; 75] years. Group 2 (a comparison group) included 610 adult hematology patients with IA (median age, 45 [18; 79] years; 42% women). A prospective case-control study was conducted to identify risk factors for IA in patients with RD: 36 rheumatic patients without IA (median age, 58 (18–79) years; 61% women) (a control group).Results and discussion. Patients with RD were found to often develop IA in the presence of anti-neutrophilic cytoplasmic antibody-associated vasculitis (granulomatosis with polyangiitis and microscopic polyangiitis) and systemic lupus erythematosus (50 and 16%, respectively). It was shown for the first time that the likelihood of IA in patients with RD increases with prolonged (median 14 days) lymphocytopenia during RD treatment (odds ratio 13.0; 95% confidence interval, 3.3–50.3). The main causative agents of IA were A. fumigatus (50%) and A. niger (29%). IA was more severe in Group 1 than in Group 2: in the resuscitation and intensive care units, there were 44 and 18%, respectively (p=0.01). Group 1 versus Group 2 more frequently had respiratory failure (61 and 37%, respectively; p=0.03), hemoptysis (28 and 7%; p=0.0001), chest pain (17 and 7%; p=0.04), and cardiac involvement (11 and 1%; p=0.0001), and less frequently had fever (67 and 85%; p=0.01). The common site of IA was the lung (83%); the characteristic feature detected by computed tomography (CT) is pulmonary cavitation (44%). Antifungal therapy was used in 89% of Group 1 patients; the overall 12-week survival was 69%.Conclusion. In patients with RD, it is difficult to differentiate between the progression of the underlying disease, adverse drug reactions, infectious complications, or a combination of these disorders due to the similarity of their clinical manifestations. When RD patients with infectious syndrome and respiratory failure develop prolonged lymphocytopenia during combination therapy, AI should be suspected and lung CT, bronchoscopy, and mycological examination of the material obtained by bronchoalveolar lavage be done.Цель исследования – изучение факторов риска развития, этиологии, особенностей клинических проявлений и эффективности лечения инвазивного аспергиллеза (ИА) у пациентов с ревматическими заболеваниями (РЗ).Пациенты и методы. Проведено первое исследование «доказанного» и «вероятного» (EORTС/MSGERC, 2019) ИА у 18 пациентов с РЗ, которые составили 3% всех взрослых больных ИА (n=699), включенных в регистр, созданный на базе кафедры клинической микологии, аллергологии и иммунологии ФГБОУ ВО «Северо-Западный государственный медицинский университет им. И.И. Мечникова» (1998–2020 гг.) – 1-я группа. В этой группе было 56% женщин, медиана возраста – 59 [21; 75] лет. Во 2-ю группу (группа сравнения) вошли 610 взрослых пациентов с гематологическими заболеваниями, страдающих ИА (медиана возраста – 45 [18; 79] лет, женщины – 42%). Для изучения факторов риска ИА у пациентов с РЗ было проведено проспективное исследование случай-контроль, в которое включили 36 взрослых пациентов с РЗ без ИА (медиана возраста – 58 [18; 79] лет, женщины – 61%) – контрольная группа.Результаты и обсуждение. Установлено, что у пациентов с РЗ ИА чаще развивается на фоне васкулитов, ассоциированных c антинейтрофильными цитоплазматическими антителами (гранулематоз с полиангиитом и микроскопический полиангиит), и системной красной волчанки (соответственно в 50 и 16% случаев). Впервые было показано, что вероятность возникновения ИА у пациентов с РЗ повышается при длительной (медиана – 14 дней) лимфоцитопении на фоне лечения РЗ (отношение шансов 13,0; 95% доверительный интервал 3,3–50,3). Основными возбудителями ИА были A. fumigatus (50% случаев) и A. niger (29%). В 1-й группе ИА протекал более тяжело, чем во 2-й: в отделениях реанимации и интенсивной терапии находились 44 и 18% больных соответственно (p=0,01). В 1-й группе чаще, чем во 2-й, отмечались дыхательная недостаточность (61 и 37% соответственно; p=0,03), кровохарканье (28 и 7%; p=0,0001), боль в грудной клетке (17 и 7%; p=0,04), поражение сердца (11 и 1%; p=0,0001), реже – лихорадка (67 и 85%; p=0,01). Основной локализацией ИА были легкие (у 83% больных), характерный признак, определяемый при компьютерной томографии (КТ), – наличие полостей деструкции (44% случаев). Антифунгальную терапию получали 89% больных 1-й группы, общая выживаемость в течение 12 нед составила 69%.Заключение. У пациентов с РЗ сложно провести дифференциальную диагностику между прогрессированием основного заболевания, неблагоприятными реакциями лекарственной терапии, инфекционным осложнением или комбинацией этих нарушений из-за сходства их клинических проявлений. При развитии длительной лимфоцитопении на фоне комплексной терапии у пациентов с РЗ, имеющих инфекционный синдром и дыхательную недостаточность, следует заподозрить ИА и провести обследование: КТ легких, бронхоскопию и микологическое исследование материала, полученного при бронхоальвеолярном лаваже.

α-Galactosidase of Aspergillus niger: purification and properties

Highly purified α-galactosidase (specific activiys 25 U/mg protein) has been isolated from the cultural liquid of Aspergillus niger. The enzyme is thermo- and pH-stable, with pH-optimum 4.1, and temperature optimum 60 °C. Km and Vmax for nitrophenyl substrate was shown to be 1.19 mM and 25 µmol/min/mg protein, respectively. α-Galactosidase was inhibited by the product of reaction D-galactose (Ki – 6.2 × 10-2M). The enzyme displayed narrow specificity towards glycon. It proved to be metal-independent. The active center of the enzyme contains the carboxylic group of the C-terminal aminoacid and imidazole group of histidine

Stability of native and modified α-galactosidase of Cladosporium cladosporioides

By modifying carbohydrate component of glycoproteins it is possible to elucidate its role in manifestation of structural and functional properties of the enzyme. The comparison of activity and stability of the native and modified by oxidation with sodium periodate α-galactosidase of Cladosporium cladosporioides was carried out. To determine α-galactosidase activity the authors used n-nitrophenyl synthetic substrate, as well as melibiose, raffinose and stachyose. Modification of the carbohydrate component had a significant effect on catalytic properties of the enzyme. Both the reduction of Vmax and enzyme affinity for natural and synthetic substrates were observed. The native enzyme retained more than 50% of the maximum activity in the range of 20-60 °C, while for the modified enzyme under the same conditions that temperature range was 30-50 °C. The modified α-galactosidase demonstrated a higher thermal stability under neutral pH conditions. The residual activity of the modified α-galactosidase was about 30% when treated with 70% (v/v) methanol, ethanol and propanol. About 50% of initial activity was observed when 40% ethanol and propanol, and 50% methanol were used. It was shown that the modification of C. cladosporioides α-galactosidase by sodium periodate is accompanied by a significant decrease in enzyme activity and stability, probably caused by topological changes in the tertiary and quaternary structure of the protein molecule

Influence of chemical reagents and UV irradiation on the activity of Penicillium canescens α-galactosidase

Investigations of the influence of chemical and physical factors on the conformational and functional properties of enzymes make a significant contribution to the study of the mechanism of action of industrially important proteins. The aim of the work was to evaluate the effect of chemical reagents and UV irradiation on the catalytic properties of Penicillium canescens α-galactosidase. Enzyme activity was assessed with p-nitrophenyl-α-D-galactopyranoside. Studies of the functionally active glycosidase groups were carried out on the basis of inhibitory and kinetic analysis using Dixon and Luinuiver-Burke methods with help of specific chemical reagents. A significant decrease in the activity of α-galactosidase in the presence of carbodiimi­des, diethylpyrocarbonate, the reagents on sulfhydryl groups was shown. A UV-induced decrease in enzyme activi­ty in the dose range of 900-7200 J/m2 was noted. Based on the data obtained, the imidazole group of histidine, carboxyl groups of C-terminal amino acids and the SH-groups of cysteine are assumed to play an important role in the manifestation of the activity of P. canescens α-galactosidase

Thermal stability of Cryptococcus albidus α-L-rhamnosidase

Yeast as well as micromycetes α-L-rhamnosidases, currently, are the most promising group of enzymes. Improving of the thermal stability of the enzyme preparation are especially important studies. Increase in stability and efficiency of substrate hydrolysis by α-L-rhamnosidase will improve the production technology of juices and wines. The aim of our study was to investigate the rate of naringin hydrolysis by α-L-rhamnosidase from Cryptococcus albidus, and also some aspects of the thermal denaturation and stabilization of this enzyme. We investigated two forms of α-L-rhamnosidase from C. albidus, which were obtained by cultivation of the producer on two carbon sources – naringin and rhamnose. A comparative study of properties and the process of thermal inactivation of α-L-rhamnosidases showed that the inducer of synthesis had no effect on the efficiency of naringin hydrolysis by the enzyme, but modified thermal stability of the protein molecule. Hydrophobic interactions and the cysteine residues are involved in maintaining of active conformation of the α-L-rhamnosidase molecule. Yeast α-L-rhamnosidase is also stabilized by 0.5% bovine serum albumin and 0.25% glutaraldehyde