Background. Neutrophil extracellular traps (NETs) are net-like structures that have been investigated in inflammatory diseases. However, the presence of NETs in infected persons without clinical symptoms has not been yet studied.Aim. To reveal NETs in healthy persons during and after the H1N1 influenza pandemic as well as to study the functional activity of NETs.Materials and methods. The study included two groups of volunteers (n = 10 in each group) aged 20–25 years. The first group of volunteers was examined in the absence of acute diseases during one month before the study and in the absence of chronic diseases in the medical history. Volunteers of the second group were in contact with patients with influenza, but did not get sick. The comparative study also included patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, abscess; 12 patients) and 9 patients with non-specific ulcerative colitis. Neutrophils were isolated from the blood by the traditional method of Ficoll density centrifugation. The number, morphology, and functional activity of NETs were determined (by capture of Klebsiella pneumoniae). SYBR Green I-based fluorescence microscopy was used to visualize and quantify NETs.Results. In healthy volunteers who were not in contact with infected patients, spontaneous NETs formation did not occur. Neutrophils of persons who were in contact with infected patients spontaneously formed NETs. In this case the number of NETs reached 8.58 ± 0.51%, and the size of NETs amounted to 39.68 ± 3.52 µm. NETs effectively captured cells of the tested microorganism, which was accompanied by retraction of network fibers and transformation of the network structure into a cloud-like one, which retained 89.38 ± 5.86 microbial cells. For comparison, the NETs in patients with acute inflammation in the abdominal cavity captured and bound 20.2 ± 1 .67 microbial cells and with non-specific ulcerative colitis – 5.53 ± 0.34 cells.Conclusion. High binding capacity of NETs is a factor contributing to effective defense of the body against the development of an infectious disease with manifested clinical symptoms.

One of the important problems of medicine is the search for new reliable markers of inflammation that would allow us to determine the type of inflammatory process. The aim of the study was to identify specific markers of infectious and aseptic inflammation based on the analysis of morphological variants of neutrophil extracellular traps (NETs). The study included 26 patients with various nosological forms of inflammation in the abdominal cavity (12 with acute appendicitis, 8 with acute cholecystitis, 6 with a diagnosis of pancreatitis/ pancreonecrosis) after surgery. The study also included 20 patients with post-COVID and a group of 10 volunteers no clinical manifestations of the disease, but at the same time had contacts with flu patients. Neutrophils were isolated using gradient centrifugation. Fluorescence microscopies with the dye SYBR Green (JSC “Evrogen”, Russia) were used for NET visualization and counting. NETs in the morphological form of neutrophils web structure were found in the blood of surgical patients with acute appendicitis who had a favorable course of the postoperative period. In a group of volunteers who were exposed to influenza but did not have symptoms of an infectious disease, we also found neutrophils web structure. In all patients with abdominal inflammation complicated by abdominal abscess, in addition to neutrophil web structure, abnormal forms of NETs were recorded, in particular, NETs in the form of single filaments. In the post-COVID patients an absolute absence of neutrophilis web structure was revealed. In all post-COVID patients, NETs are found only in the form of single filaments. The results of subsequent experiments showed that collagen peptides are inducers of the formation of NETs in the form of single filaments. The detection of neutrophils web structure is a sign of an infectious inflammatory process. NETs in the morphological form of single filaments are markers of aseptic inflammation, as well as a sign of endothelial damage.

Background. Many studies have shown that neutrophil extracellular traps (NETs) in the form of web-like structures are present in the peripheral blood of patients with inflammatory diseases. In our research, in addition to traditional web-like NET structures, several anomalous forms were identified, including NETs with cloud-like appearance.Aim. To investigate morphological and functional transformation of NETs under the influence of Klebsiella pneumoniae and immunoglobulin G (IgG).Materials and methods. The study included 42 patients of Moscow City Clinical Hospital No. 51: 28 patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, pancreatitis, peritonitis), 6 patients diagnosed with ulcerative colitis, and 8 patients with hernias. Neutrophils were isolated using gradient-density centrifugation. To calculate NETs, we used SYBR Green I-induced fluorescence microscopy (Evrogen, Russia), with the dye specifically interacting with double-stranded DNA. The functional activity of NETs was determined in the Klebsiella pneumoniae (ATCC 700603) capture test.Results. In patients with inflammatory diseases of the abdominal cavity in the postoperative period, the functional activity of NETs was several times lower than in healthy individuals. NETs in these patients capture and bind no more than 20 cells of the microorganism. Under the effect of IgG, neutrophil networks transform into loose cloud-like structures, which can hardly capture and bind the pathogen, binding only 8.46 ± 0.44 cells of the microorganism. Spontaneous enzymatic degradation of cloud like NETs may be accompanied by the production of secondary alteration factors.Conclusion. The results of the study provide the grounds for the development of new approaches to elaborating vaccination regimens and using immunobiologics that require preliminary monitoring of the state of innate immunity, in particular, neutrophil networks in the patient’s body.

Развитие инфекционных осложнений у больных с воспалительными заболеваниями определяется тем, насколько эффективную защитную реакцию нейтрофилы больного будут развивать при контакте с патогеном. Противоинфекционная устойчивость организма во многом определяется готовностью нейтрофилов формировать нейтрофильные сети, способные эффективно захватывать патогены. Цель исследования. Определение параметров нейтрофильных экстраклеточных ловушек (НЭЛ) у больных с воспалительными заболеваниями брюшной полости. Методика. Обследованы 28 прооперированных больных с различными формами абдоминального воспаления и 6 терапевтических больных с язвенным колитом, 6 пациентов обследованы при поступлении. Нейтрофилы выделяли, используя градиентное центрифугирование. Для подсчета НЭЛ использовали флюоресцентную микроскопию с красителем SYBR Green (Evrogen; Россия), специфично взаимодействующего с двухцепочечной ДНК. Функциональную активность НЭЛ определяли в тесте с захватом Klebsiella pneumoniae (ATCC 700603). Результаты. При исследовании больных в послеоперационном периоде обнаружили, что при остром аппендиците/абсцессе, остром холецистите и остром панкреатите/панкреонекрозе обнаруживаются близкие количества НЭЛ – 12,63±2,13%, 15,66±2,81% и 12,64±2,73%. В этих же группах больных регистрируются достоверные различия в размерах НЭЛ, которые достигают 46,82±6,70 мкм, 73,62±16,29 мкм и 96,84±27,17 мкм, соответственно. Увеличение размеров НЭЛ в этих группах больных объясняется присутствием, помимо сетевидных структур, дополнительно еще и нитевидных, волокнистых и вуалеобразных нейтрофильных внеклеточных структур. Группа больных с неспецифическим язвенным колитом, получающих консервативное лечение (группа сравнения), демонстрирует тенденцию к уменьшению размеров НЭЛ – до величины 31,96±14,75 мкм. Исследование функциональной активности НЭЛ показывает, что её снижение предшествует развитию септического состояния (на примере выявленного клинического случая). Заключение. Все, без исключения, исследованные нозологические формы воспаления в брюшной полости сопровождаются формированием НЭЛ в виде нейтрофильных сетей. Помимо нейтрофильных сетей воспаление индуцирует у части больных формирование НЭЛ в виде одиночных нитей, волокон и вуалей. Количественные параметры НЭЛ коррелируют с размерами и распространением очага воспаления на соседние анатомические области. На примере описанного клинического случая обнаружено резкое ослабление функциональной активности НЭЛ, которое предшествовало развитию септического осложнения. Прослеживается связь между исходно ослабленной функциональной активностью НЭЛ и развитием септического осложнения у пациента в послеоперационном периоде. The development of infectious complications in patients with inflammatory diseases is determined by how effective protective reaction the patient’s neutrophils will develop upon contact with the pathogen. The body’s anti-infective resistance is largely determined by the willingness of neutrophils to form neutrophilic networks capable of effectively capturing pathogens. Aim. Determination of parameters of neutrophil extracellular traps (NETs) in patients with inflammatory diseases of the abdominal cavity. Methods. 28 operated patients with various forms of abdominal inflammation and 6 therapeutic patients with ulcerative colitis were examined, 6 patients were examined upon admission. Neutrophils were isolated using gradient centrifugation. To calculate the NETs, fluorescence microscopy with the dye SYBR Green (Evrogen; Russia), which specifically interacts with double-stranded DNA, was used. The functional activity of NETs was determined in the Klebsiella pneumoniae capture test (ATCC 700603). Results. In the study of patients in the postoperative period, it was found that in acute appendicitis/abscess, acute cholecystitis and acute pancreatitis/pancreonecrosis, similar amounts of NETs are found – 12,63±2,13%, 15,66±2,81% and 12.64±2.73%. In the same groups of patients, significant differences in the size of the NETs are recorded, which reach 46.82±6.70 microns, 73.62±16.29 microns and 96.84±27.17 microns, respectively. The increase in the size of the NETs in these groups of patients is explained by the presence, in addition to network-like structures, additionally filamentous, fibrous and cloud-like neutrophil extracellular structures. A group of patients with nonspecific ulcerative colitis receiving conservative treatment (comparison group) shows a tendency to decrease the size of the NEL to a value of 31.96±14.75 microns. A study of the functional activity of NETs shows that its decrease precedes the development of a septic condition (using the example of an identified clinical case). Conclusion. All, without exception, the studied nosological forms of inflammation in the abdominal cavity are accompanied by the formation of NETs in the form of neutrophilic networks. In addition to neutrophilic networks, inflammation induces the formation of NETs in the form of single strands, fibers and clouds form in some patients. The quantitative parameters of the NETs correlate with the size and spread of the inflammatory focus to neighboring anatomical areas. On the example of the described clinical case, a sharp decrease in the functional activity of NETs was found, which preceded the development of a septic complication. There is a connection between the initially weakened functional activity of NETs and the development of septic complications in the patient in the postoperative period.

Background. Neutrophil extracellular traps (NETs) are net-like structures that have been investigated in inflammatory diseases. However, the presence of NETs in infected persons without clinical symptoms has not been yet studied. Aim. To reveal NETs in healthy persons during and after the H1N1 influenza pandemic as well as to study the functional activity of NETs. Materials and methods. The study included two groups of volunteers (n = 10 in each group) aged 20-25 years. The first group of volunteers was examined in the absence of acute diseases during one month before the study and in the absence of chronic diseases in the medical history. Volunteers of the second group were in contact with patients with influenza, but did not get sick. The comparative study also included patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, abscess; 12 patients) and 9 patients with non-specific ulcerative colitis. Neutrophils were isolated from the blood by the traditional method of Ficoll density centrifugation. The number, morphology, and functional activity of NETs were determined (by capture of Klebsiella pneumoniae). SYBR Green I-based fluorescence microscopy was used to visualize and quantify NETs. Results. In healthy volunteers who were not in contact with infected patients, spontaneous NETs formation did not occur. Neutrophils of persons who were in contact with infected patients spontaneously formed NETs. In this case the number of NETs reached 8.58 +/- 0.51%, and the size of NETs amounted to 39.68 +/- 3.52 mu m. NETs effectively captured cells of the tested microorganism, which was accompanied by retraction of network fibers and transformation of the network structure into a cloud-like one, which retained 89.38 +/- 5.86 microbial cells. For comparison, the NETs in patients with acute inflammation in the abdominal cavity captured and bound 20.2 +/- 1 .67 microbial cells and with non-specific ulcerative colitis - 5.53 +/- 0.34 cells. Conclusion. High binding capacity of NETs is a factor contributing to effective defense of the body against the development of an infectious disease with manifested clinical symptoms.

We studied the neutrophils and monocytes obtained from 37 patients with various inflammatory diseases such as psoriasis, acute infectious process in the abdominal cavity (acute appendicitis/abscess of the abdominal cavity, and acute cholecystitis), acute pancreatitis, and post-COVID syndrome after mild COVID infection. The number and the morphological structure of neutrophil extracellular traps (NET) as well as the effect of IgG on NET were examined. NET were visualized and counted by fluorescence microscopy with fluorescent dye SYBR Green. All the studied types of inflammation were accompanied by spontaneous formation of NET. After application of IgG, the number of NET doubled, their size increased, and transformation of net-like traps into the cloud forms was observed. The clouds form structure of the network is not capable of capturing pathogens with subsequent retraction, the products of its enzymatic degradation can be the factors of secondary alteration. The study results demonstrate a previously unknown mechanism of infection resistance.