Typhoidal salmonella disease in Mukuru informal settlement, Nairobi Kenya; carriage, diversity, and antimicrobial resistant genes.

INTRODUCTION: Multiple studies have shown that typhoid fever is endemic in developing countries characterized by poor hygiene. A unique way of Salmonella Typhi (S.Typhi) pathogenicity is establishing a persistent, usually asymptomatic carrier state in some infected individuals who excrete large numbers of bacteria in faeces. This study aimed to determine the isolation rate of S.Typhi from blood and stool samples among cases and asymptomatic individuals in the Mukuru informal settlement and identify antibiotic resistance patterns within the same population. MATERIALS AND METHODS: We recruited 1014 outpatient participants presenting with typhoid-like symptoms in selected health centres in Nairobi, Kenya. Bacterial isolation was done on Xylose Lysine Deoxycholate agar (XLD) and Mac Conkey agar (Oxoid), followed by standard biochemical tests. Identification was done using API20E, and S.Typhi was confirmed by serotyping using polyvalent antisera 0-9 and monovalent antisera d. The Kirby-Bauer disc diffusion method was used to test the antimicrobial susceptibility of S.Typhi isolates, while Multi-Drug Resistant (MDR) strains were characterized using conventional PCR. RESULTS: Of 1014 participants, 54 (5%) tested positive for S.Typhi. Thirty-eight (70%) of the S.Typhi isolated were from stool samples, while sixteen (30%) were from blood. Three (0.2%) of the isolates were from asymptomatic carriers. Of the 54 S.Typhi isolates, 20 (37%) were MDR. Resistance to ciprofloxacin and nalidixic acid was 43% and 52%, respectively. Resistance to amoxicillin-clavulanic acid (a beta-lactam inhibitor) was 2%. The BlaTEM-1 gene was present in 19/20 (95%) MDR isolates. CONCLUSION: MDR S.Typhi is prevalent in Mukuru Informal settlement. The sharp increase in nalidixic acid resistance is an indication of reduced susceptibility to fluoroquinolones, which are currently the recommended drugs for the treatment of typhoid fever. This study highlights the need for effective antimicrobial stewardship and routine surveillance of antimicrobial resistance (AMR) to inform policy on the prevention and control of MDR Typhoid disease.

IRAS/Nischarin modulates morphine reward by glutamate receptor activation in the nucleus accumbens of mouse brain.

The I1 imidazoline receptor and its candidate protein imidazoline receptor antisera-selected (IRAS)/Nischarin are linked to µ opioid receptor (MOR) functions associated with MOR trafficking. We previously demonstrated that IRAS may play an important role in the development of morphine tolerance and physical dependence in vivo. However, the effects of IRAS on morphine psychological dependence are not fully understood. To extend these studies, we investigated the impact of IRAS on morphine dependence in conditioned place preference (CPP) experiments and explored the underlying mechanisms. Knockout of IRAS enhanced the acquisition and reinstatement of morphine-induced CPP. Conditional-knockout of IRAS in the nucleus accumbens (NAc) reproduced higher CPP, and overexpression of IRAS in the NAc rescued the increased morphine-induced CPP in IRAS-/- mice. IRAS-/- mice showed dramatic cAMP-dependent protein kinase (PKA) activation, upregulation of the phosphorylation of the AMPA receptor GluR1-S845 and NMDA receptor NR1-S897 in the NAc after CPP experiment. Moreover, knockout of IRAS induced an increase in spontaneous excitatory postsynaptic current (sEPSC) frequency and a decrease in the AMPA/NMDA ratio in the NAc after chronic morphine treatment. The selective AMPA receptor antagonist NBQX could inhibit morphine CPP in WT mice, while its effect was significantly reduced in IRAS-/- mice. Together, our results demonstrate that IRAS contributes to the regulation of morphine dependence and that the alteration of glutamatergic transmission in the NAc may participate in the effect of IRAS.

Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.

G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site-deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site-specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.

History repeats itself: horse originated hyperimmune sera production against SARS CoV-2

Background/aim/AIM: SARS-CoV-2 disease was announced as a pandemic by The World Health Organization in early 2020. It is still threatening the world population. Here, we aimed to produce hyperimmune sera that contain immunoglobulin G and F(ab')2 fragments sourced from horse antibodies as an urgent response to the pandemic. Materials and methods: SARS-CoV-2 was produced and inactivated with three different methods [formaldehyde (FA), formaldehyde, and binary ethylene amine (FA + BEI), and heat treatment]. After in vitro inactivation control, immunogens were mixed with Freund's adjuvant, thereafter horses (n: 2 for FA, 4 for FA + BEI, 2 for heat inactivation) and New Zealand rabbits (n: 6 for FA, 6 fo r FA + BEI, 6 for heat inactivation) were immunized four times. Neutralizing antibody levels of the sera were measured at the 4th, 6th, and 8th weeks. When the antibodies were detected at the peak level, plasma was collected from horses and hyperimmune sera procured after the purification process. Results: Horses and rabbits produced highly neutralizing antibodies against the SARS-CoV-2 in FA and FA + BEI inactivation groups, foreign proteins were removed effectively after purification. Conclusion: This study presents a profitable practice to develop specific antisera in horses against SARS-CoV-2 for emergency and low-cost response. In further studies, new purification methods can be used to increase the efficiency of the final product.

The high-affinity immunoglobulin receptor FcγRI potentiates HIV-1 neutralization via antibodies against the gp41 N-heptad repeat.

The HIV-1 gp41 N-heptad repeat (NHR) region of the prehairpin intermediate, which is transiently exposed during HIV-1 viral membrane fusion, is a validated clinical target in humans and is inhibited by the Food and Drug Administration (FDA)-approved drug enfuvirtide. However, vaccine candidates targeting the NHR have yielded only modest neutralization activities in animals; this inhibition has been largely restricted to tier-1 viruses, which are most sensitive to neutralization by sera from HIV-1-infected individuals. Here, we show that the neutralization activity of the well-characterized NHR-targeting antibody D5 is potentiated >5,000-fold in TZM-bl cells expressing FcγRI compared with those without, resulting in neutralization of many tier-2 viruses (which are less susceptible to neutralization by sera from HIV-1-infected individuals and are the target of current antibody-based vaccine efforts). Further, antisera from guinea pigs immunized with the NHR-based vaccine candidate (ccIZN36)3 neutralized tier-2 viruses from multiple clades in an FcγRI-dependent manner. As FcγRI is expressed on macrophages and dendritic cells, which are present at mucosal surfaces and are implicated in the early establishment of HIV-1 infection following sexual transmission, these results may be important in the development of a prophylactic HIV-1 vaccine.

Immunotherapy targeting the Streptococcus pyogenes M protein or streptolysin O to treat or prevent influenza A superinfection.

Viral infections complicated by a bacterial infection are typically referred to as coinfections or superinfections. Streptococcus pyogenes, the group A streptococcus (GAS), is not the most common bacteria associated with influenza A virus (IAV) superinfections but did cause significant mortality during the 2009 influenza pandemic even though all isolates are susceptible to penicillin. One approach to improve the outcome of these infections is to use passive immunization targeting GAS. To test this idea, we assessed the efficacy of passive immunotherapy using antisera against either the streptococcal M protein or streptolysin O (SLO) in a murine model of IAV-GAS superinfection. Prophylactic treatment of mice with antiserum to either SLO or the M protein decreased morbidity compared to mice treated with non-immune sera; however, neither significantly decreased mortality. Therapeutic use of antisera to SLO decreased morbidity compared to mice treated with non-immune sera but neither antisera significantly reduced mortality. Overall, the results suggest that further development of antibodies targeting the M protein or SLO may be a useful adjunct in the treatment of invasive GAS diseases, including IAV-GAS superinfections, which may be particularly important during influenza pandemics.

Immune serum-activated human macrophages coordinate with eosinophils to immobilize Ascaris suum larvae.

Helminth infection represents a major health problem causing approximately 5 million disability-adjusted life years worldwide. Concerns that repeated anti-helminthic treatment may lead to drug resistance render it important that vaccines are developed but will require increased understanding of the immune-mediated cellular and antibody responses to helminth infection. IL-4 or antibody-activated murine macrophages are known to immobilize parasitic nematode larvae, but few studies have addressed whether this is translatable to human macrophages. In the current study, we investigated the capacity of human macrophages to recognize and attack larval stages of Ascaris suum, a natural porcine parasite that is genetically similar to the human helminth Ascaris lumbricoides. Human macrophages were able to adhere to and trap A suum larvae in the presence of either human or pig serum containing Ascaris-specific antibodies and other factors. Gene expression analysis of serum-activated macrophages revealed that CCL24, a potent eosinophil attractant, was the most upregulated gene following culture with A suum larvae in vitro, and human eosinophils displayed even greater ability to adhere to, and trap, A suum larvae. These data suggest that immune serum-activated macrophages can recruit eosinophils to the site of infection, where they act in concert to immobilize tissue-migrating Ascaris larvae.

Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation.

This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.

Induction of superovulation using inhibin antiserum and competence of embryo development in wild large Japanese field mice (Apodemus speciosus).

Seasonally, bred wild mice provide a unique bioresource, with high genetic diversity that differs from wild-derived mice and laboratory mice. This study aimed to establish an alternative superovulation method using wild large Japanese field mice (Apodemus speciosus) as the model species. Specifically, we investigated how the application of inhibin antiserum and equine chorionic gonadotropin (IASe) during both the reproductive and non-reproductive seasons impact the ovulation rate and competence of embryo development after in vitro fertilization (IVF) with fresh and cryopreserved sperm. When the wild mice were superovulated by injecting eCG followed by human chorionic gonadotropin (hCG), few oocytes were collected during the reproductive and non-reproductive seasons. In comparison, the number of ovulated oocytes was dramatically enhanced by the administration of IASe, followed by isolation of ovulated oocytes 24 hr after 30 IU hCG administration. The IVF oocytes that were in vitro cultured (IVC) with medium containing serum further developed to the 2- and/or 4-cell stage using both fresh and frozen-thawed sperm. In conclusion, we successfully established an alternative protocol for collecting ovulated oocytes from wild large Japanese field mice by administering IASe and hCG during both the reproductive and non-reproductive seasons. This study is the first to develop IVF-IVC wild large Japanese field mice beyond the 2- and/or 4-cell stage in vitro using fresh and cryopreserved sperm. This approach could be used in other species of wild or endangered mice to reduce the number of animals used for experiments, or in maintaining stocks of germ cells or embryos.

A combination of antibodies against Bm86 and Subolesin inhibits engorgement of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae in vitro.

BACKGROUND: Rhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro. RESULTS: Antiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium. CONCLUSIONS: Combining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

Fluorine-modified sialyl-Tn-CRM197 vaccine elicits a robust immune response.

Even though a vaccine that targets tumor-associated carbohydrate antigens on epithelial carcinoma cells presents an attractive therapeutic approach, relatively poor immunogenicity limits its development. In this study, we investigated the immunological activity of a fluoro-substituted Sialyl-Tn (F-STn) analogue coupled to the non-toxic cross-reactive material of diphtheria toxin197 (CRM197). Our results indicate that F-STn-CRM197 promotes a greater immunogenicity than non-fluorinated STn-CRM197. In the presence or absence of adjuvant, F-STn-CRM197 remarkably enhances both cellular and humoral immunity against STn by increasing antigen-specific lymphocyte proliferation and inducing a mixed Th1/Th2 response leading to production of IFN-γ and IL-4 cytokines, as well as STn-specific antibodies. Furthermore, antisera produced from F-STn-CRM197 immunization significantly recognizes STn-positive tumor cells and increases cancer cell lysis induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) pathways. Our data suggest that this F-STn vaccine may be useful for cancer immunotherapy and possibly for prophylactic prevention of cancer.

Ovulation of juvenile, mature, and aged female C57BL/6 mice following coadministration of inhibin antiserum and equine chorionic gonadotropin.

Superovulation technique is important to improve the efficiency of oocyte and animal production and reduce the number of oocyte donors. Previously, we have reported that the coadministration of inhibin antiserum (IAS) and equine chorionic gonadotropin (eCG) results in the production of >100 oocytes in a 4-week-old female C57BL/6 mice. It is well established that superovulation depends on the age of the female mice. However, detailed data regarding the ovulation of juvenile, mature, and aged female mice following the administration of IAS and eCG as well as the performance of reproductive technologies using oocytes have not yet been investigated. In the present study, we examined the effect of the age of female mice (3-50 weeks old) on the number of ovulated oocytes via the coadministration of IAS and eCG or eCG alone. Treatment with IAS plus eCG produced the maximum number of oocytes at 4 weeks of age. Moreover, IAS plus eCG produced more oocytes than eCG alone in mice aged between 3 and 5 weeks or 7 and 30 weeks. The fertilization and birth rates were similar between the two treatments at any age. Moreover, after vitrifying and warming the embryos, the survival and birth rates of two-cell embryos were similar between the two treatments. Subsequently, we examined the optimal ages of female mice (between 24 and 34 days) to obtain a high and stable number of oocytes. In mice aged between 24 and 32 days, IAS plus eCG induced the production of more eggs than eCG alone. Notably, the coadministration of IAS and eCG in mice aged between 25 and 31 days resulted in stable ovulation and high number of oocytes. Using the tip of the optimal female aged between 25 and 31 days old, we demonstrated an efficient production of embryos and offspring between homozygous knockout males and few females aged 26-28 days via in-vitro fertilization and embryo transfer. In summary, the coadministration of IAS and eCG resulted in a higher number of oocytes in juvenile, mature, and aged female mice. This treatment may be useful for the efficient production of homozygous mutant mice from a limited number of female mice.

Joint treatment with azidothymidine and antiserum for eradication of avian leukosis virus subgroup a contamination in vaccine virus seeds.

Contamination of avian attenuated vaccines by avian leukosis virus (ALV) is considered to be a specific horizontal transmission of ALV. Eradication of ALV contamination in vaccine virus seeds is thus a precondition of qualified vaccine production. In this study, we used the nucleoside reverse transcriptase (RT) inhibitor azidothymidine (AZT) together with monofactor antiserum against avian leukosis virus subgroup A (ALV-A) to remove ALV-A from vaccine virus seeds. Different doses of ALV-A were artificially added to the Newcastle disease virus (NDV) vaccine seeds, then the ALV-contaminated attenuated virus vaccine virus seeds were cultured in DF-1 cells. Single-drug treatment with 5 µg/mL AZT or 5% (v/v) as well as combined treatment with AZT and antiserum significantly suppressed ALV-A replication (P < 0.001) in the vaccine virus seeds. Complete absence of virus replication was observed in cells exposed to joint treatment with AZT and antiserum. The treated virus seeds met the requirements of the Chinese Ministry of Agriculture. Therefore, combined treatment with AZT and antiserum can be used to eradicate contaminating ALV-A from vaccine virus seeds, thus providing a new approach for improving vaccine safety.

Variability in venom composition of European viper subspecies limits the cross-effectiveness of antivenoms.

Medically relevant cases of snakebite in Europe are predominately caused by European vipers of the genus Vipera. Systemic envenoming by European vipers can cause severe pathology in humans and different clinical manifestations are associated with different members of this genus. The most representative vipers in Europe are V. aspis and V. berus and neurological symptoms have been reported in humans envenomed by the former but not by the latter species. In this study we determined the toxicological profile of V. aspis and V. berus venoms in vivo in mice and we tested the effectiveness of two antivenoms, commonly used as antidotes, in counteracting the specific activities of the two venoms. We found that V. aspis, but not V. berus, is neurotoxic and that this effect is due to the degeneration of peripheral nerve terminals at the NMJ and is not neutralized by the two tested antisera. Differently, V. berus causes a haemorrhagic effect, which is efficiently contrasted by the same antivenoms. These results indicate that the effectiveness of different antisera is strongly influenced by the variable composition of the venoms and reinforce the arguments supporting the use polyvalent antivenoms.

A bivalent protein r-PB, comprising PA and BclA immunodominant regions for comprehensive protection against Bacillus anthracis.

Anthrax infection is primarily initiated by B. anthracis endospores that on entry into the host germinate to vegetative cells and cause severe bacteremia and toxaemia employing an array of host colonisation factors and the lethal tripartite toxin. The protective efficacy of conventional protective antigen (PA) based anthrax vaccines is improved by co-administration with inactivated spores or its components. In the present study, using structural vaccinology rationale we synthesized a bivalent protein r-PB encompassing toxin (PAIV) and spore components (BclACTD) and characterized its protective efficacy against B. anthracis infection. Active immunization of mice with r-PB generated high titer circulating antibodies which facilitated the phagocytic uptake of spores, inhibited their germination to vegetative cells and completely neutralized anthrax toxins in vivo resulting in 100 % survival against anthrax toxin challenge. Proliferation of CD4+ T cell subsets with up-regulation of Th1 (IFN-γ, IL-2, and IL-12), Th2 (IL-5, IL-10) cytokines and balanced expression of IgG1:IgG2a antibody isotypes indicated the stimulation of both Th1 and Th2 subsets. The immunized mice exhibited 100 % survival upon challenge with B. anthracis spores or toxin indicating the ability of r-PB to provide comprehensive protection against anthrax. Our results thus demonstrate r-PB an efficient vaccine candidate against anthrax infection.

Carrier Cells for Delivery of Oncolytic Measles Virus into Tumors: Determinants of Efficient Loading.

Oncolytic measles virus (OMV) is a promising antitumor agent. However, the presence of anti-measles neutralizing antibodies (NAbs) against the hemagglutinin (H) protein of OMV is a major barrier to the therapeutic application of OMV in clinical practice. In order to overcome this challenge, specific types of cells have been used as carriers for OMV. Differential loading strategies appear to result in different therapeutic outcomes; despite this, only few studies have reported practical ex vivo loading strategies required for effective treatment. To this end, we systematically evaluated the antitumor efficacy of OMV using different loading strategies; this involved varying the in vitro loading duration and loading dose of OMV. We found that improved oncolysis of carrier cells was achieved by a prolonged loading duration in the absence of NAbs. However, the enhanced oncolytic effect was abrogated in the presence of NAbs. Further, we found that the expression of H protein on the surface of carrier cells was predominantly determined by the loading duration rather than the loading dose. Finally, we showed that NAbs blocked viral transfer by targeting H protein prior to the occurrence of cell-to-cell interactions. Our results provide comprehensive information on the determinants of an effective loading strategy for carrier cell-based virotherapy; these results may be useful for guiding the application of OMV as an antitumor agent in clinical practice.

Role of Endogenous Melatonin in the Regulation of Th17/Treg Balance during Pregnancy.

We studied the role of endogenous melatonin in the development and functioning of T cells that produce IL-17 (Th17) and regulatory T cells (Treg) during pregnancy. The study was performed ex vivo and in vitro with auto-serum as the source of endogenous melatonin under conditions of blockade of melatonin-dependent signaling. Participation of the hormone in the regulation of differentiation of both CD4+RORγt+ and CD4+FoxP3+T cells and their key products IL-17A and TGF-ß was demonstrated. It is known that the normal gestational process is accompanied by a decrease in Th17/Treg ratio due to hormonal changes. The sensitivity of the studied subpopulations to melatonin during pregnancy can affect its outcome.

Dynamics of formation and morphological features of neutrophil extracellular traps formed under the influence of opsonized Staphylococcus aureus.

In the process of performing their protective functions, neutrophils can form neutrophil extracellular traps (NETs), consisting of DNA in combination with enzymes and histones. The aim of the study was to determine the dynamics of the formation of NETs under the influence of opsonized Staphylococcus aureus and to determine the morphological features of their development in real time by atomic force microscopy. It was found that the maximum formation of NETs was observed after 3 hours of co-incubation of neutrophils and opsonized S. aureus. For the first time, the atomic force microscopy method revealed that, at first, large blocks of parallel DNA helices are formed, which then spread in waves, and only then their bifurcation and separation can be observed. Some of the strands formed are covered by a shell, which subsequently completely disappears. Enzymes and histones become clearly visible only after 140 to 150 minutes of observation. The DNA helixes move toward the opsonized S. aureus. After NET formation, the cell remains on the substrate only in the form of traces of focal adhesion. This, and the fact that the maximum amount of NETs is formed after 3 hours of co-incubation with opsonized S. aureus, suggests that the formation of NETs follows the classical mechanism. The study of the dynamics of formation and the microstructure of NETs makes it possible to estimate the time frame for the implementation of this protective mechanism of the human body when performing the compensatory inflammatory reaction.