RESUMO
It has long been known that containers for sample analysis or storage can play a role in endotoxin recovery and have to be taken into account when determining endotoxin concentrations. However, there is little data on the effects of containers regarding (1â3)-ß-D-glucan, which plays a role as a contaminant in endotoxin measurements. To determine the effect of the container on (1â3)-ß-D-glucan measurements, four different types of containers were investigated at different temperatures and stored for up to 28 days. For short-term storage for 3 h at room temperature, no effect of the container on the (1â3)-ß-D-glucan recovery could be observed, but for storage at -20 °C, the results indicate that the storage time and temperature influences (1â3)-ß-D-glucan detection. All containers showed a trend of lower recoveries over time, but the polyethylene container showed a significantly lower recovery compared to the other containers. We also showed that freeze/thaw cycles had a strong influence on the recovery of (1â3)-ß-D-glucan in polyethylene containers. Our study showed that the container can affect not only the detection of endotoxins but also the detection of (1â3)-ß-D-glucans.
Assuntos
Glucanos , beta-Glucanas , Glucanos/análise , beta-Glucanas/análise , Endotoxinas , Temperatura , PolietilenosRESUMO
Among the mechanisms put-up by the host to defend against invading microorganisms, antimicrobial peptides represent the first line. In different species of mammals, the cathelicidin family of antimicrobial peptides AMPs has been identified, and in humans, LL-37 is the only type of cathelicidin identified. LL-37 has many different biological activities, such as regulation of responses to inflammation, besides its lipopolysaccharide (LPS)-neutralizing and antimicrobial and activities. Recently, employing a murine septic model that involves cecal ligation and puncture (CLP), we examined the effect of LL-37. The results indicated that LL-37 exhibits multiple protective actions on septic mice; firstly, the survival of CLP mice was found to be improved by LL-37 by the suppression of the macrophage pyroptosis that induces the release of pro-inflammatory cytokines (such as IL-1ß) and augments inflammatory reactions in sepsis; secondly, the release of neutrophil extracellular traps (NETs), which have potent bactericidal activity, is enhanced by LL-37, and protects mice from CLP-induced sepsis; thirdly, LL-37 stimulates neutrophils to release antimicrobial microvesicles (ectosomes), which improve the pathological condition of sepsis. These findings indicate that LL-37 protects CLP septic mice through at least three mechanisms, i.e., the suppression of pro-inflammatory macrophage pyroptosis and the release of antimicrobial NETs (induction of NETosis) and ectosomes from neutrophils. Thus, LL-37 can be a potential therapeutic candidate for sepsis due to its multiple properties, including the modulation of cell death (pyroptosis and NETosis) and the release of antimicrobial NETs and ectosomes as well as its own bactericidal and LPS-neutralizing activities.
Assuntos
Antibacterianos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Sepse/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Piroptose , CatelicidinasRESUMO
Endotoxins are cell wall components of Gram-negative bacteria. A release of endotoxins into the human blood stream results in an inflammation reaction that can lead to life-threatening conditions like sepsis. Therefore, control for endotoxin contamination of intravenously administered drugs is crucial. Drugs are usually tested for putative endotoxin contamination with Limulus-based tests. However, validity of the compendial test procedures is questioned in the case of low endotoxin recovery (LER). To assure validity, regulatory authorities request hold-time studies of endotoxin in addition to pharmacopoeial requirements. Within these studies, endotoxin is added (spiked) to an undiluted product. The spiked product is held for a certain period of time and subsequently diluted for endotoxin determination. Due to the known heterogeneity of endotoxin the question has been raised as to which source represents the most adequate endotoxin spike. In the present study, endotoxin hold-time studies were analyzed by using different sources of endotoxin. Highly purified endotoxin, crude endotoxin extracts (Naturally Occurring Endotoxin) from different bacterial species and varied growth conditions as well as endogenous endotoxin contaminations were investigated. The results clearly demonstrate that endotoxin masking-an effect of LER-is dependent on the endotoxin source used. Various parameters such as bacterial strain and growth conditions lead to different masking susceptibilities. Due to these effects it is impossible to predict the susceptibility of bacterial endotoxin contamination to LER. In order to determine whether a sample is prone to LER, an endotoxin spike that is susceptible to LER is required.
Assuntos
Endotoxinas/metabolismo , Bactérias Gram-Negativas/crescimento & desenvolvimento , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/metabolismo , Fatores de TempoRESUMO
The inability to detect endotoxin added to undiluted drug samples has been called: Low Endotoxin Recovery (LER). The phenomenon has caused concerns amongst drug manufacturing quality control scientists in that manufactured solutions contaminated with endotoxin could show false-negative results via routine Limulus-based tests. The time-dependent appearance of LER has been analyzed in detail to provide a better understanding of the mechanism. The assumption has been that the root-cause of LER involves the interplay of endotoxin with surfactants and results in aggregate structures that are complexed with surfactants. The endotoxin molecules when complexed with surfactants are not accessible for Limulus-based detection. The results demonstrate a predominant role of complex-forming agents. It was shown that although the presence of surfactants is a strong prerequisite for masking, it does not determine the kinetics of endotoxin masking. Interestingly, the endotoxin concentration itself had no substantial impact on LER kinetics. By adjusting the ratios of complex-forming constituents, including surfactant, chelator and endotoxin, and by testing the order in which the constituents are added, a new model for simulating masking kinetics has been determined. Our work provides for the first time a model to simulate masking kinetics of endotoxin which lends a better understanding of LER.
Assuntos
Endotoxinas/análise , Teste do Limulus/métodos , Preparações Farmacêuticas/análise , Quelantes/química , Humanos , Controle de Qualidade , Tensoativos/químicaRESUMO
LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 suppresses the LPS/ATP-induced pyroptosis of macrophages in vitro by both neutralizing the action of LPS and inhibiting the response of P2X7 (a nucleotide receptor) to ATP. Thus, in this study, we further evaluated the effect of LL-37 on pyroptosis in vivo using a cecal ligation and puncture (CLP) sepsis model. As a result, the intravenous administration of LL-37 improved the survival of the CLP septic mice. Interestingly, LL-37 inhibited the CLP-induced caspase-1 activation and pyroptosis of peritoneal macrophages. Moreover, LL-37 modulated the levels of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) in both peritoneal fluids and sera, and suppressed the activation of peritoneal macrophages (as evidenced by the increase in the intracellular levels of IL-1ß, IL-6 and TNF-α). Finally, LL-37 reduced the bacterial burdens in both peritoneal fluids and blood samples. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the pyroptosis of macrophages, and inflammatory cytokine production by activated macrophages and bacterial growth. Thus, the present findings imply that LL-37 can be a promising candidate for sepsis because of its many functions, such as the inhibition of pyroptosis, modulation of inflammatory cytokine production and antimicrobial activity.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Citocinas/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Sepse/tratamento farmacológico , Sepse/microbiologia , Animais , Lipopolissacarídeos/toxicidade , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Sepse/imunologia , Sepse/patologia , CatelicidinasRESUMO
Over the last few decades Limulus Amebocyte Lysate (LAL) has been the most sensitive method for the detection of endotoxins (Lipopolysaccharides) and is well accepted in a broad field of applications. Recently, Low Endotoxin Recovery (LER) in biopharmaceutical drug products has been noticed, whereby the detection of potential endotoxin contaminations is not ensured. Notably, most of these drug products contain surfactants, which can have crucial effects on the detectability of endotoxin. In order to analyze the driving forces of LER, endotoxin detection in samples containing nonionic surfactants in various buffer systems was investigated. The results show that the process of LER is kinetically controlled and temperature-dependent. Furthermore, only the simultaneous presence of nonionic surfactants and components capable of forming metal complexes resulted in LER. In addition, capacity experiments show that even hazardous amounts of endotoxin can remain undetectable within such formulation compositions. In conclusion, the LER phenomenon is caused by endotoxin masking and not by test interference. In this process, the supramolecular structure of endotoxin is altered and exhibits only a limited susceptibility in binding to the Factor C of Limulus-based detection systems. We propose a two-step mechanism of endotoxin masking by complex forming agents and nonionic surfactants.
Assuntos
Teste do Limulus/métodos , Lipopolissacarídeos/análise , Tensoativos/química , Lipopolissacarídeos/químicaRESUMO
Endotoxins are a highly pyrogenic and immunogenic contaminant of bacterial origin that must be avoided during the manufacturing of biopharmaceutical products to ensure safety and efficacy. Low endotoxin recovery, also known as a masking effect, is defined as the ability to detect <50% [21] of the expected endotoxin in an endotoxin assay. Masking can be caused by the ability of endotoxins to build aggregates, bind to the protein or organise in micelles or vesicles that in turn inhibit detection of the endotoxin in the solution being tested. Therefore, a masking effect can result from physical parameters of the molecule being tested or from the buffer/environmental conditions of the solution the molecule is in. This can subsequently lead to the underestimation of endotoxin contaminations and lead to a potential false negative test. Tight control over the effectiveness of the downstream process and the use of well-characterised endotoxin testing assays are needed to ensure optimal endotoxin removal. This manuscript demonstrates the capacity to remove the endotoxins within a proven acceptable range by also controlling and evaluating the potential masking effects during downstream process at ambient temperature and also during sample storage condition until the analyse was performed. The endotoxin removal study (ERS) is divided in the initial part to evaluate the process buffers and the conditions of the molecule to avoid the underestimation of endotoxins in process samples in advance. This pre-study is a necessary prerequisite to evaluate the results after the endotoxin spiked downstream unit operations. With those aspects, the removal capacity can be demonstrated. A study was carried out to characterise the endotoxin removal capability of the purification process including controlling of masking effects. The endotoxin removal capacity on ion exchange chromatography and during ultrafiltration/diafiltration unit operations of the downstream processing of an immunoglobulin G1 antibody was conducted using various process parameters to understand their impact on endotoxin removal. In the small-scale study, the processing steps from each tested unit operation were spiked with Escherichia coli endotoxins. The potential masking effect during purification was addressed by controlling the hold time by spiking studies of the different generated pools at ambient temperature. By conducting a masking study, all generated protein pools (flow-through/wash, eluate and regeneration pools) had no masking effect caused through sample handling prior to analysis. Overall, this study showed that endotoxins could be successfully removed by anion exchange chromatography. A partial removal could be achieved by cation exchange chromatography and endotoxins could not be removed with ultrafiltration/diafiltration.
Assuntos
Produtos Biológicos , Cromatografia por Troca Iônica , Endotoxinas , ProteínasRESUMO
The blue blood of the horseshoe crab is a natural, irreplaceable, and precious resource that is highly valued by the biomedical industry. The Limulus amebocyte lysate (LAL) obtained from horseshoe crab blood cells functions as a surprisingly sophisticated sensing system that allows for the extremely sensitive detection of bacterial and fungal cell-wall components. Notably, LAL tests have markedly contributed to the quality control of pharmaceutical drugs and medical devices as successful alternatives to the rabbit pyrogen test. Furthermore, LAL-based endotoxin and (1â3)-ß-D-glucan (ß-glucan) assay techniques are expected to have optimal use as effective biomarkers, serving as adjuncts in the diagnosis of bacterial sepsis and fungal infections. The innovative ß-glucan assay has substantially contributed to the early diagnosis and management of invasive fungal diseases; however, the clinical significance of the endotoxin assay remains unclear and is challenging to elucidate. Many obstacles need to be overcome to enhance the analytical sensitivity and clinical performance of the LAL assay in detecting circulating levels of endotoxin in human blood. Additionally, there are complex interactions between endotoxin molecules and blood components that are attributable to the unique physicochemical properties of lipopolysaccharide (LPS). In this regard, while exploring the potential of new LPS-sensing technologies, a novel platform for the ultrasensitive detection of blood endotoxin will enable a reappraisal of the LAL assay for the highly sensitive and reliable detection of endotoxemia.
RESUMO
Monocyte activation tests (MAT) are widely available but rarely used in place of animal-based pyrogen tests for safety assessment of medical devices. To address this issue, the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the PETA International Science Consortium Ltd. convened a workshop at the National Institutes of Health on September 18-19, 2018. Participants included representatives from MAT testing laboratories, medical device manufacturers, the U.S. Food and Drug Administration's Center for Devices and Radiologic Health (CDRH), the U.S. Pharmacopeia, the International Organization for Standardization, and experts in the development of MAT protocols. Discussions covered industry experiences with the MAT, remaining challenges, and how CDRH's Medical Device Development Tools (MDDT) Program, which qualifies tools for use in evaluating medical devices to streamline device development and regulatory evaluation, could be a pathway to qualify the use of MAT in place of the rabbit pyrogen test and the limulus amebocyte lysate test for medical device testing. Workshop outcomes and follow-up activities are discussed.
Assuntos
Equipamentos e Provisões/efeitos adversos , Monócitos/fisiologia , Testes de Toxicidade/métodos , Alternativas aos Testes com Animais , Animais , Endotoxinas , Pirogênios , CoelhosRESUMO
Endotoxin (lipopolysaccharide) testing of drugs is routinely required in pharmaceutical industries. Suitable compendial assays are defined by national pharmacopoeias. At this time, Limulus Amoebocyte Lysate (LAL) assays are the gold standard. LAL is used in vitro for specific detection of endotoxin based on endotoxin-activated Factor C-mediated clotting cascade. However, alternative mediated pathways (e.g., Factor G), impurities, and further factors may influence test results. Some of these influencing factors are eliminated by recombinant Factor C (rFC) test, which represents a promising alternative. rFC not only enables highly specific endotoxin testing, as interfering Horseshoe Crab blood components are eliminated, but also offers ethical and ecological advantages compared to classical LAL assays. However, the question remains whether rFC-based tests are robust test systems, equivalent or superior to LAL and suitable for routine bacterial endotoxin testing. Pharmaceutical test users have validated the test successfully for their specific products, but no long-term studies have been published that combine testing of unknown samples, inter-laboratory, -operator, and -lot changes. Thus, it was of great interest to investigate rFC test performance in a routine setting within a proficiency test program set-up. Over a period of six years comparative endotoxin testing was conducted with one kinetic chromogenic LAL assay and two rFC-based assays. Results of this study demonstrate that both rFC-based assays were comparable to LAL. All results met acceptance criteria defined by compendial bacterial endotoxin testing. RFC-based methods generated results with even better endotoxin recovery rates compared to LAL. Therefore, rFC-based tests were found to represent reliable methods, as equivalent or even superior to LAL assays and suitable for routine bacterial endotoxin testing.
RESUMO
Sepsis is a life-threatening disease caused by systemic dys-regulated inflammatory response to infection. We previously revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of cecal ligation and puncture septic mice. Ectosomes, microvesicles released from neutrophils, are reported to be elevated in sepsis survivors; however, the functions of ectosomes in sepsis remain largely unknown. Therefore, we herein elucidated the protective action of LL-37 on sepsis, by focusing on LL-37-induced ectosome release in a cecal ligation and puncture model. The results demonstrated the enhancement of ectosome levels by LL-37 administration, accompanied by a reduction of bacterial load. Importantly, ectosomes isolated from LL-37-injected cecal ligation and puncture mice contained higher amounts of antimicrobial proteins/peptides and exhibited higher antibacterial activity, compared with those from PBS-injected cecal ligation and puncture mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. Actually, LL-37 stimulated mouse bone-marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed antibacterial potential. Furthermore, administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of cecal ligation and puncture mice. Together these observations suggest LL-37 induces the release of antimicrobial ectosomes in cecal ligation and puncture mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Micropartículas Derivadas de Células/metabolismo , Neutrófilos/metabolismo , Sepse/imunologia , Animais , Carga Bacteriana , Ceco/cirurgia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Ativação de Neutrófilo , CatelicidinasRESUMO
For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in "Guidance for Industry: Pyrogen and Endotoxins Testing." It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.
RESUMO
LL37 is the sole antimicrobial peptide of human cathelicidin comprising 37 amino acids, which is expressed mainly in epithelial cells and neutrophils, and activates mast cells. In the present study, in order to elucidate the mechanism of mast cell activation by LL37, the associations between the internalization of LL37 and Masrelated gene X2 (MrgX2)mediated mast cell activation (degranulation) was investigated using the human mast cell line, LAD2. LL37 was rapidly internalized into the cells, and induced degranulation, as assessed by the extracellular release of ßhexosaminidase. Pertussis toxin, a Gprotein inhibitor, significantly suppressed the internalization of LL37 and the degranulation of LAD2 cells. Furthermore, small interfering (si)RNAmediated knockdown of MrgX2, a putative G proteincoupled receptor for LL37, inhibited the internalization of LL37 and degranulation of LAD2 cells. Notably, LL37 internalization was enhanced by the stable expression of MrgX2 in HMC1 and 293 cells. In addition, the internalized LL37 mainly colocalized with MrgX2 in the perinuclear region of LAD2 cells. Furthermore, neuraminidase treatment, which removes negatively charged sialic acid from the cell surface, markedly reduced the internalization of LL37 and degranulation of LAD2 cells, and clathrinmediated endocytosis inhibitors (dynasore and chlorpromazine) inhibited the internalization and degranulation of LAD2 cells. Taken together, these observations indicated that LL37 may bind the negatively charged cell surface molecules, rapidly internalize into the cells via clathrinmediated endocytosis and interact with MrgX2 to activate mast cells (LAD2 cells).
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Degranulação Celular/genética , Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Neuropeptídeos/genética , Linhagem Celular , Células Cultivadas , Endocitose , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , CatelicidinasRESUMO
Upon exposure to invading microorganisms, neutrophils undergo NETosis, a recently identified type of programmed cell death, and release neutrophil extracellular traps (NETs). NETs are described as an antimicrobial mechanism, based on the fact that NETs can trap microorganisms and exhibit bactericidal activity through the action of NETassociated components. In contrast, the components of NETs have been recognized as damageassociated molecular pattern molecules (DAMPs), which trigger inflammatory signals to induce cell death, inflammation and organ failure. In the present study, to clarify the effect of NETs on cytokine production by macrophages, mouse macrophagelike J774 cells were treated with NETs in combination with lipopolysaccharide (LPS) as a constituent of pathogenassociated molecular patterns. The results revealed that NETs significantly induced the production of interleukin (IL)1ß by J774 cells in the presence of LPS. Notably, the NET/LPSinduced IL1ß production was inhibited by both caspase1 and caspase8 inhibitors. Furthermore, nucleases and serine protease inhibitors but not antihistone antibodies significantly inhibited the NET/LPSinduced IL1ß production. Moreover, we confirmed that caspase1 and caspase8 were activated by NETs/LPS, and the combination of LPS, DNA and neutrophil elastase induced IL1ß production in reconstitution experiments. These observations indicate that NETs induce the production of IL1ß by J774 macrophages in combination with LPS via the caspase1 and caspase8 pathways, and NETassociated DNA and serine proteases are involved in NET/LPSinduced IL1ß production as essential components.
Assuntos
Armadilhas Extracelulares/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Animais , Células CHO , Caspase 1/metabolismo , Caspase 8/metabolismo , Cricetulus , DNA/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Peroxidase/metabolismo , Serina Proteases/metabolismo , Transdução de SinaisRESUMO
LL37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL37 may modulate various inflammatory reactions. The authors previously revealed that LL37 improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. In the present study, the mechanism for the protective action of LL37 was elucidated using the CLP model, focusing on the effect of LL37 on the release of neutrophil extracellular traps (NETs). The results indicated that the intravenous administration of LL37 suppressed the increase of damage-associated molecular patterns (DAMPs), including histoneDNA complex and highmobility group protein 1, in addition to interleukin1ß, tumor necrosisα and soluble triggering receptor expressed on myeloid cells (TREM)1 in plasma and peritoneal fluids. Notably, LL37 significantly suppressed the decrease of mononuclear cell number in blood, and the increase of polymorphonuclear cell (neutrophil) number in the peritoneal cavity during sepsis. Furthermore, LL37 reduced the bacterial burden in blood and peritoneal fluids. Notably, LL37 increased the level of NETs (myeloperoxidaseDNA complex) in plasma and peritoneal fluids. In addition, it was verified that LL37 induces the release of NETs from neutrophils, and NETs possess the bactericidal activity. Overall, these observations suggest that LL37 improves the survival of CLP septic mice by possibly suppressing the inflammatory responses as evidenced by the inhibition of the increase of cytokines, soluble TREM1 and DAMPs (host cell death) and the alteration of inflammatory cell numbers, and bacterial growth via the release of NETs with bactericidal activity.