Post-COVID syndrome is characterized by fatigue, reduced exercise tolerance, muscle and joint pain, and psychoemotional disorders. In the development of a generalized body response in a viral infection, abnormal defense responses are of great importance. We studied neutrophils, neutrophil extracellular traps (NETs), DNA degradation products (purine nitrogenous bases, PNBs), and traditional biochemical parameters.Aim. To determine biochemical parameters and the number of NETs and PNBs in the peripheral blood of patients with post-COVID syndrome.Materials and methods. The study included outpatients (n = 21) aged 18–59 years (36 [27 ÷ 50]). The control group consisted of 20 individuals aged 18–59 years (38.5 [29 ÷ 51.5]) without a past medical history of the coronavirus infection. All patients underwent a physical examination, their medical history was assessed, and the level of NETs and PNBs in the venous blood was determined.Results. 11 patients had a mild form of the disease in their past medical history, 7 – moderate, and 3 – severe. The most common symptoms in the patients were fatigue, headache, epigastric pain, dizziness, and joint pain. Hair loss and dyspnea were less common. The concentration of NETs and PNBs was higher in the patients with post-COVID syndrome than in the control group (p < 0.05). We detected NETs in the patients with post-COVID syndrome only in the form of filamentous structures. The concentration of extracellular purine bases in the blood of the patients with post-COVID syndrome was the highest in patients with moderate and severe acute periods. In patients with a mild acute period, the concentration of PNBs was 7.38 [0.0 ÷ 60.7] mg / ml, and in patients with moderate and severe acute periods – 19.15 [0.0 ÷ 33.5] and 34.19 [3.35 ÷ 70.0] mg / ml, respectively.Conclusion. Extracellular purine bases in concentrations capable of causing secondary alteration of cells are found in the peripheral blood of patients with post-COVID syndrome. Post-COVID syndrome is accompanied by the formation of filamentous NETs in the blood of patients.
Нейтрофильные экстраклеточные ловушки (НЭЛ) возникают в результате высвобождения гранулярного и ядерного содержимого нейтрофилов во внеклеточное пространство в ответ на различные классы микроорганизмов, растворимые факторы и собственные модифицированные антигены организма. Во многих исследованиях продемонстрировано, что образование НЭЛ является эффективным механизмом борьбы с внедряющимися микроорганизмами, поскольку недостаточность формирования НЭЛ или гидролиз основной нуклеотидной цепи НЭЛ бактериальными ДНКазами делает организм человека восприимчивым к инфекциям. Основная роль НЭЛ - предотвращение распространения микроорганизмов в организме. Принято считать, что образование нейтрофильных экстраклеточных ловушек должно строго регулироваться, чтобы избежать повреждения тканей и избыточной гемокоагуляции. Цель исследования - морфологическая характеристика основных типов структур нейтрофильных экстраклеточных ловушек в зависимости от вида воспалительного процесса. Методика. В исследование были включены 18 больных с различными видами воспаления (абсцесс брюшной полости, аппендицит, панкреонекроз, калькулезный холецистит) в острый период заболевания с высокими показателями лейкоцитоза (10-12 тыс/мкл). Для визуализации и подсчета нейтрофильных экстраклеточных ловушек использовали метод флуоресцентной микроскопии. Окрашивание НЭЛ проводили с помощью флюоресцентного красителя для двухцепочечной ДНК SYBR Green (Evrogen). Результаты. Показано, что при благоприятном течении острого инфекционного процесса нейтрофилы выбрасывают одну или несколько нитей c ДНК, которые затем ветвятся и формируют структуру сети. В дальнейшем происходит ретракция волокон сети с захватом микроорганизмов. При постнекротическом воспалении сеть не формируется, а вместо неё возникает вуалеобразная структура, состоящая из тонких нитей c ДНК. Асептическое воспаление характеризуется особой морфологической формой -- нитевидной. Нейтрофил, при этом виде воспаления, выбрасывает значительной длины одиночную нить c ДНК. Заключение. Предложенный нами метод визуализации нативных нейтрофильных экстраклеточных ловушек показал высокую диагностическую эффективность. Он позволяет выявлять различия в структурах нейтрофильных экстраклеточных ловушек при разных типах воспаления. Neutrophil extracellular traps (NETs) arise as a result of the release of granular and nuclear contents of neutrophils into the extracellular space in response to various classes of microorganisms, soluble factors and own modified antigens. Many studies have demonstrated that the formation of NETs is an effective mechanism for combating invading microorganisms, since insufficient release of NETs or hydrolysis of the main nucleotide chain of NET by bacterial DNases increases susceptibility to infections. The main role of NET is to prevent the spread of microorganisms. It is generally believed that the formation of NETs should be strictly regulated to avoid tissue damage and excessive hemocoagulation. The aim of the study was to identify the main morphological structures of NETs depending on the type of inflammatory process. Methods. The study included 18 patients with various types of inflammation (abdominal abscess, appendicitis, pancreatic necrosis, calculous cholecystitis) in the acute period of the disease with high rates of leukocytosis (10-12 103/µl). NETs were stained with a fluorescent dye for double-stranded DNA, SYBR Green (Evrogen), and visualized and counted using fluorescence microscopy. Results. The study showed that, with a benign course of an acute infectious process, neutrophils emit one or more DNA strands, which then branch and form the network structure. In the future, the fibers of the network retract, capturing microorganisms. With necrotic inflammation, the network is not formed, but instead a veil-like structure consisting of thin strands of DNA appears. Aseptic inflammation is characterized by a special morphological form, i.e., a threadlike form. Neutrophils, with this type of inflammation, emit a single strand with DNA of considerable length. Conclusion. Thus, our proposed method of visualization of native NETs has shown high efficiency. It allowed us to identify different structures of NETs in various types of inflammation (infectious, necrotic and aseptic), which seems relevant and opens up a new direction in pathophysiology of inflammation.
Introduction. Interferon-containing drugs are frequently used, but their effect on the innate immune cells response and adaptive immunity parameters is not well known.Aim. To investigate the effect of interferon-alpha-containing drugs on the production of neutrophil extracellular traps, the population and subpopulation composition of peripheral blood lymphocytes in cell culture conditions.Materials and methods. We used peripheral blood from 12 healthy donors and 6 patients with acute inflammation, which was used as a neutrophil and lymphocyte source. Neutrophil extracellular traps were induced by two methods: either spontaneously, without stimulation, or by lipopolysaccharides.Results and discussions. The investigation of innate immunity responses showed that neutrophils from healthy donors demonstrated a slight spontaneous production of neutrophil extracellular traps during incubation (4 h). The significant NET increase was observed after stimulation with lipopolysaccharides up to 31.59 ± 2.32% after 2-hour incubation period, and up to 42.93 ± 3.56% after 4-hour incubation period. Viferon does not have a significant effect on the number of neutrophilic extracellular traps, but significantly increases their size. Kipferon limits the excessive production of neutrophil extracellular traps, reducing the number of these structures, and also significantly reduces their size and changes their morphology. Ergoferon causes not only a rapid increase in the number of neutrophilic extracellular traps, but also significantly changes their morphology. The extremely long DNA fibers that go beyond the scope of view are observed when exposed to Ergoferon. Kipferon stimulates the production of several morphological forms of neutrophilic extracellular traps at once.Conclusion. The development of innate immune responses is mainly maintained by Viferon and Kipferon. At the same time, Kipferon restrains the intensity of the inflammatory reaction and increases the number of active NK cells. The conducted multilevel study allow researchers to identify new properties and mechanisms of action of administered pharmacological products containing interferon-α, which confirm the effective stimulation of individual components of the immune system due to their action.
Post-COVID syndrome (long covid, post COVID-19 condition) is characterized by cognitive and mental disorders, chest and joint pain, impaired sense of smell and taste, as well as by gastrointestinal and cardiac disorders. The diagnosis of post-COVID syndrome is based mainly on the patients' complaints. To date, no optimal diagnostic method has been proposed. The study was aimed to compare the informative value of the indicators obtained during conventional assessment of patients with post-COVID syndrome and the blood levels of neutrophil (NETs) and monocyte (METs) extracellular traps. The study involved neutropils and monocytes collected from 21 patients with post-COVID syndrome aged 18–59. Fluorescence microscopy and the SYBR Green (Evrogen) fluorescent dye for double-stranded DNA were used for enumeration and imaging of extracellular traps. Clinical and laboratory indicators make it impossible to identify the changes specific for post-COVID syndrome. At the same time, post-COVID syndrome is characterized by inflammation in the vascular endothelium. The filamentous forms of NETs found in blood are a laboratory feature of such aseptic inflammation. The filamentous forms of NETs have been detected only in those patients who have a history of mild to severe СOVID-19, while the filamentous forms of METs have been found in patients having a history of severe infection. The findings show that the detection of the filamentous forms of NETs and METs in blood is the most informative diagnostic feature of post-COVID syndrome.
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