Neutrophil Necrosis Research Articles

Development and Validation of an Individualized Immune Prognostic Signature in Early-Stage Nonsquamous Non–Small Cell Lung Cancer

The prevalence of early-stage non-small cell lung cancer (NSCLC) is expected to increase with recent implementation of annual screening programs. Reliable prognostic biomarkers are needed to identify patients at a high risk for recurrence to guide adjuvant therapy. To develop a robust, individualized immune signature that can estimate prognosis in patients with early-stage nonsquamous NSCLC. This retrospective study analyzed the gene expression profiles of frozen tumor tissue samples from 19 public NSCLC cohorts, including 18 microarray data sets and 1 RNA-Seq data set for The Cancer Genome Atlas (TCGA) lung adenocarcinoma cohort. Only patients with nonsquamous NSCLC with clinical annotation were included. Samples were from 2414 patients with nonsquamous NSCLC, divided into a meta-training cohort (729 patients), meta-testing cohort (716 patients), and 3 independent validation cohorts (439, 323, and 207 patients). All patients underwent surgery with a negative surgical margin, received no adjuvant or neoadjuvant therapy, and had publicly available gene expression data and survival information. Data were collected from July 22 through September 8, 2016. Overall survival. Of 2414 patients (1205 men [50%], 1111 women [46%], and 98 of unknown sex [4%]; median age [range], 64 [15-90] years), a prognostic immune signature of 25 gene pairs consisting of 40 unique genes was constructed using the meta-training data set. In the meta-testing and validation cohorts, the immune signature significantly stratified patients into high- vs low-risk groups in terms of overall survival across and within subpopulations with stage I, IA, IB, or II disease and remained as an independent prognostic factor in multivariate analyses (hazard ratio range, 1.72 [95% CI, 1.26-2.33; P < .001] to 2.36 [95% CI, 1.47-3.79; P < .001]) after adjusting for clinical and pathologic factors. Several biological processes, including chemotaxis, were enriched among genes in the immune signature. The percentage of neutrophil infiltration (5.6% vs 1.8%) and necrosis (4.6% vs 1.5%) was significantly higher in the high-risk immune group compared with the low-risk groups in TCGA data set (P < .003). The immune signature achieved a higher accuracy (mean concordance index [C-index], 0.64) than 2 commercialized multigene signatures (mean C-index, 0.53 and 0.61) for estimation of survival in comparable validation cohorts. When integrated with clinical characteristics such as age and stage, the composite clinical and immune signature showed improved prognostic accuracy in all validation data sets relative to molecular signatures alone (mean C-index, 0.70 vs 0.63) and another commercialized clinical-molecular signature (mean C-index, 0.68 vs 0.65). The proposed clinical-immune signature is a promising biomarker for estimating overall survival in nonsquamous NSCLC, including early-stage disease. Prospective studies are needed to test the clinical utility of the biomarker in individualized management of nonsquamous NSCLC.

Morphine worsens the severity and prevents pancreatic regeneration in mouse models of acute pancreatitis

BackgroundOpioids such as morphine are widely used for the management of pain associated with acute pancreatitis. Interestingly, opioids are also known to affect the immune system and modulate inflammatory pathways...

The critical role of myeloperoxidase in Streptococcus pneumoniae clearance and tissue damage during mouse acute otitis media.

We have recently reported that neutrophils play a pivotal role in innate defense against Streptococcus pneumoniae ( Spn) during mouse acute otitis media (AOM). However, the underlying mechanism remains unclear. By constructing models of pneumococcal AOM in C57BL/6 mice and using a specific inhibitor invivo, we investigated the role of myeloperoxidase (MPO), one of the most important protein components of neutrophils. Experiment results showed a significant increase in MPO production of the recruited neutrophils in Spn-infected mice. Neutrophils killed Spn in a MPO-dependent manner. MPO facilitated the generation of reactive oxygen species (ROS), and consequently promoted Spn clearance at an early stage and exacerbated tissue damage. Moreover, MPO induced neutrophil apoptosis and necrosis, which, in turn, worsened tissue damage. In summary, our study demonstrates that neutrophil MPO plays a paradoxical role in bacterial clearance and tissue damage in pneumococcal AOM.

The role of nitric oxide in apoptosis modulation in newborns with pneumonia

Introduction. Nitric oxide (NO) is an im-portant diagnostic marker and mediator in the inflammatory process, which plays a key role in the mechanism of programmed cell death, thus, forming the basis of many pathological diseases.Methods. The study involved 73 newborns with pneumonia (moderate severity in 44 neonates (group 1), severe pneumonia in 29 (group 2)). The intensity of neutrophil apoptosis and necrosis was determined by flow cytometry, whereas nitric oxide me-tabolites were measured by spectropho-tometry.&nbsp;Results. The level of nitric oxide metabo-lites (NO2+NO3) in newborns with pneu-monia was higher than in healthy children (16.93 (15.82; 17.79) μmol/ml) and cor-related with disease severity (in group 1 – 22.65 (21.42; 23.40) μmol/ml in group 2 – 26.82 (25.81; 27.91) μmol/ml). The level of NO3 increased moderately, while NO2 generation was more intense, exceed-ing control indexes in both groups (рc-1&lt;0.001; рc-2&lt;0.001; р1-2&lt;0.001).The occurrence of intensive neutrophil apoptosis was revealed in newborns with pneumonia of moderate severity (рc-1&lt;0.001), while necrosis prevailed in se-vere pneumonia (рc-2&lt;0.001).Inverse correlation (R=-0.63; р&lt;0.05) was found between the level of nitric oxide metabolites and&nbsp;neutrophil apoptosis; and direct correlation (R=0.68; р&lt;0.05) was re-vealed between NO metabolites and neu-trophil necrosis indices.Conclusions. Increased generation of nitric oxide metabolites, that directly correlated with disease severity in newborns with pneumonia, was found. NO2 has multidi-rectional effects on neutrophil apoptosis and necrosis, leading to toxic accumula-tion of neutrophils in the organism, thus enhancing the inflammatory and intoxica-tion process that impact disease severity.

Galectin‑9 ameliorates fulminant liver injury.

Fulminant hepatitis is a severe liver disease resulting in hepatocyte necrosis. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that has been evaluated as a potential therapeutic agent for various diseases that regulate the host immune system. Concanavalin A (ConA) injection into mice results in serious, immune-mediated liver injury similar to human viral, autoimmune and fulminant hepatitis. The present study investigated the effects of Gal-9 treatment on fulminant hepatitis in vivo and the effect on the expression of microRNAs (miRNAs), in order to identify specific miRNAs associated with the immune effects of Gal-9. A ConA-induced mouse hepatitis model was used to investigate the effects of Gal-9 treatment on overall survival rates, liver enzymes, histopathology and miRNA expression levels. Histological analyses, TUNEL assay, immunohistochemistry and miRNA expression characterization, were used to investigate the degree of necrosis, fibrosis, apoptosis and infiltration of neutrophils and macrophages. Overall survival rates following ConA administration were significantly higher in Gal-9-treated mice compared with control mice treated with ConA + PBS. Histological examination revealed that Gal-9 attenuated hepatocellular damage, reduced local neutrophil infiltration and prevented the local accumulation of macrophages and liver cell apoptosis in ConA-treated mice. In addition, various miRNAs induced by Gal-9 may contribute to its anti-apoptotic, anti-inflammatory and pro-proliferative effects on hepatocytes. The results of the present study demonstrate that Gal-9 may be a candidate therapeutic target for the treatment of fulminant hepatitis.

Milk fat globule-EGF factor 8 suppresses the aberrant immune response of systemic lupus erythematosus-derived neutrophils and associated tissue damage

Abnormal features of the systemic lupus erythematosus (SLE)-derived neutrophils, promoted aberrant immune response, have inspired new studies of the induction of autoimmunity and the development of organ damage in SLE. In this study, we explore the effect of milk fat globule-EGF factor 8 (MFG-E8) on the aberrant nitrification features in pristane-induced lupus. SLE patients and mice with pristane-induced lupus develop autoantibodies associated with MFG-E8 overproduction. However, the deletion of MFG-E8 leads to uncontrolled early pulmonary and peritoneal inflammation and tissue damage in mice with pristane-induced lupus. Consistent with these findings, MFG-E8-deficient mice that are exposed to pristane show enhanced neutrophil accumulation and increased neutrophil death, including apoptosis, necrosis and NETosis, as well as impaired phagocytosis of macrophages. The consequences are the expansion of diffuse pulmonary hemorrhage, increased anti-nuclear antibody, anti-dsDNA antibody and anti-neutrophil cytoplasmic antibody levels, and enhanced immune complexes deposition and neutrophil extracellular traps (NETs) formation in the lung and kidney tissues of MFG-E8-deficient mice exposed to pristane. In patients with SLE and mice with pristane-induced lupus, neutrophil accumulation is elevated, which depends on higher expression of the surface receptor CXCR2. After pretreatment with recombinant MFG-E8, the surface expression of CXCR2 on neutrophil is downregulated, and the MFG-E8 deletion increase CXCR2 expression by ~40%. These studies indicate that MFG-E8 reduces neutrophil migration and NETosis via downregulating surface CXCR2 expression in parallel with its role in the phagocytosis of apoptotic neutrophils, suggesting that MFG-E8 may serve as a therapeutic agent for attenuating the early inflammatory responses of SLE and protect patients from lupus-related damage.

Expression of scavenger receptor A in rat's liver tissue during acute obstructive cholangitis and its significance.

ObjectiveAcute obstructive cholangitis (AOC) is a severe disease that leads to sepsis, shock and multiple organ dysfunction syndrome. Liver is the largest and the major organ for the defense mechanisms during the sepsis. Our aim was to investigate the expression of scavenger receptor A (SRA) in rat’s liver tissue during AOC and its relation with the inflammatory mediators and hepatic injury caused by endotoxins.MethodsNinety Wistar rats were randomly divided into three equal groups. In group one, the choledochus were ligagted and Escherichia coli O111B4 injected into it to produce AOC model. Group two had choledochus ligated and group three had sham surgery. Six animals from each group were sacrificed at 0, 3, 6, 12 and 24 h for comparative analysis of endotoxins, tumor necrosis factor-alpha, interleukin-6 in plasma and expression of SRA protein and SRA mRNA in liver tissue. The P < 0.05 was considered significant. Ethical approval was obtained.ResultsThe plasma endotoxins, tumor necrosis factor-alpha and interleukin-6 levels in AOC rats increased progressively with time. The SRA protein and SRA mRNA expression decreased with time. The differences among groups were significant (P < 0.01). Liver histopathology showed gradually aggravated hepatic injury with neutrophil infiltration, degeneration and necrosis of hepatocytes.ConclusionIn AOC, the expression of SRA in liver gradually decreased with progressive increase in level of plasma endotoxins and hepatic injury suggesting its importance in the defense mechanism.

Matters of life and death. How neutrophils die or survive along NET release and is "NETosis"=necroptosis?

Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders.

When is pneumonia not pneumonia: a clinicopathologic study of the utility of lung tissue biopsies in determining the suitability of cadaveric tissue for donation.

Healthcare-associated pneumonia (HCAP) represents a major diagnostic challenge because of the relatively low sensitivity and specificity of clinical criteria, radiological findings, and microbiologic culture results. It is often difficult to distinguish between pneumonia, underlying pulmonary disease, or conditions with pulmonary complications; this is compounded by the often-subjective clinical diagnosis of pneumonia. We conducted this study to determine the utility of post-mortem lung biopsies for diagnosing pneumonia in tissue donors diagnosed with pneumonia prior to death. Subjects were deceased patients who had been hospitalized at death and diagnosed with pneumonia. Post-mortem lung biopsies were obtained from the anatomic portion of the cadaveric lung corresponding to chest radiograph abnormalities. Specimens were fixed, stained with hematoxylin and eosin, and read by a single board-certified pathologist. Histological criteria for acute pneumonia included intense neutrophilic infiltration, fibrinous exudates, cellular debris, necrosis, or bacteria in the interstitium and intra-alveolar spaces. Of 143 subjects with a diagnosis of pneumonia at time of death, 14 (9.8%) had histological evidence consistent with acute pneumonia. The most common histological diagnoses were emphysema (53%), interstitial fibrosis (40%), chronic atelectasis (36%), acute and chronic passive congestion consistent with underlying cardiomyopathy (25%), fibro-bullous disease (12%), and acute bronchitis (11%). HCAP represents a major diagnostic challenge because of the relatively low sensitivity and specificity of clinical criteria, radiological findings, and microbiologic testing. We found that attending physician-diagnosed pneumonia did not correlate with post-mortem pathological diagnosis. We conclude that histological examination of cadaveric lung tissue biopsies enables ascertainment or rule out of underlying pneumonia and prevents erroneous donor deferrals.

IL-16 and MIF: messengers beyond neutrophil cell death.

Most cytokines are synthesized de novo upon appropriate stimulation, packed into vesicles and secreted from the producing cell. However, cells can also contain preformed cytokines, which means that these cytokines are synthesized prior to activation and stored inside the cell. Harboring of preformed effector molecules is essential for cells of low transcription and translation activity, as seen in neutrophils. Being part of the first line of defense against invading pathogens, neutrophils are rapidly recruited from the circulation into the infected tissue, where they immediately execute a multitude of antimicrobial effector functions.1 Several antimicrobial effector substances are stored inside neutrophils as preformed molecules. This allows a fast action, as these preformed substances can act on demand, without the need of time-consuming de novo synthesis. The presence of preformed cytokines in neutrophils emphasizes that these cells are not solely fast-acting effector cells but they can also regulate very rapidly the inflammatory and immune responses.2 Recently, we reported the storage of IL-16 and MIF in primary human neutrophils in the new journal Cell Death Discovery.3 Analysis of neutrophil granule fractions and confocal immune fluorescent microscopy revealed that IL-16 and MIF are stored in the cytosol rather than in neutrophil granules.3 Notably, IL-16 is synthesized as a biologically inactive precursor, pre-IL-16 (formerly also termed pro-IL-16), which is processed in a caspase-3-dependent manner. The C-terminal fragment IL-16C is the biologically active, secreted pro-inflammatory cytokine.4 Intracellular proteolytic activation by cytosolic proteases has also been described for other pro-inflammatory cytokines such as IL-18, IL-1β and IL-33.5, 6 Neutrophils are short-lived cells. After undergoing apoptosis they are cleared from the circulation in liver, spleen and bone marrow. Although their life span is extended at sites of infection/inflammation, large numbers of neutrophils undergo apoptosis after executing their effector functions. Apoptotic cells and blebs maintain their membrane integrity and are cleared by phagocytes in a process called efferocytosis. Since apoptotic cells/blebs are surrounded by an intact cell membrane, this process prevents the leakage/release of potentially harmful cellular content of neutrophils into the surrounding tissue. In case of insufficient or impaired efferocytosis, however, apoptotic neutrophils undergo secondary necrosis, resulting in the leakage of cell content through the disintegrated plasma membrane. Therefore, harboring preformed biologically active molecules not only favors rapid effector functions, but also bears the risk of uncontrolled release during primary necrosis or secondary necrosis. This passive release may lead to tissue damage, but is also believed to contribute to the development of autoimmunity to/against neutrophil constituents.7 The secretion mechanism of IL-16C and MIF from neutrophils, but also other cells, remains largely elusive. As both cytokines lack the signal peptide to be transferred to the ER, they are thought to be released by unconventional secretion mechanisms, as known for IL-1 and FGF2.8, 9 Previous studies hypothesized that IL-16 and MIF could be released actively from the producing cells.10, 11 However, our data indicate that in neutrophils both cytokines are released passively after the cells undergo secondary necrosis3 (Figure 1). After the screening of a large panel of substances, among others, TLR ligands, pro-inflammatory cytokines, lipid mediators, glucocorticoids, and the bacteria Escherichia coli and Staphylococcus aureus, no release of IL-16C and MIF was observed from viable or apoptotic neutrophils.3 Instead, release of IL-16C and MIF correlated strongly with the secondary necrosis of neutrophils.3 Figure 1 Storage, processing and release of IL-16 and MIF in human neutrophils. MIF and IL-16 are preformed cytokines stored in the cytosol of human neutrophils. During apoptosis, the precursor of IL-16 (pre-IL-16) is processed into the biologically active IL-16C ... As neutrophils release IL-16C and MIF only after undergoing secondary necrosis, it is tempting to speculate that IL-16C and MIF function as danger signals in case of insufficient clearance of apoptotic neutrophils. At sites of acute infection or inflammation, efferocytosis may not be sufficient to clear the large numbers of apoptotic neutrophils. Consequently, apoptotic neutrophils undergo secondary necrosis. Although the release of IL-16C and MIF from secondary necrotic neutrophils may be required to maintain and modulate the local defense and inflammatory functions, we have to keep in mind that they may also be detrimental in conditions of autoimmunity. Neutrophils, although known for a long time and studied extensively, are more complex and sophisticated in their functions than was imaginable one or two decades ago. By the release of neutrophil extracellular traps, they can fulfill antimicrobial effector functions even beyond their death.12 The study published in Cell Death Discovery3 extends this view by demonstrating that neutrophils can fulfill even their regulatory functions beyond their life span.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol.

Neutrophils harbor a number of preformed effector proteins that allow for immediate antimicrobial functions without the need for time-consuming de novo synthesis. Evidence indicates that neutrophils also contain preformed cytokines, including interleukin (IL)-1ra, CXCL8 and CXCL2. In the search for additional preformed cytokines, a cytokine array analysis identified IL-16 and macrophage migration inhibitory factor (MIF) as preformed cytokines in lysates from human primary neutrophils. Both IL-16 and MIF are unconventional cytokines because they lack a signal sequence. Using confocal immunofluorescence microscopy as well as western blot analysis of subcellular fractions, IL-16 and MIF were found to be stored in the cytosol rather than in the granules of human neutrophils, which implies an unconventional secretion mechanism for both cytokines. IL-16 is synthesized and stored as a precursor (pre-IL-16). We present evidence that the processing of pre-IL-16 to the biologically active IL-16C is mediated by caspase-3 and occurs during both spontaneous and UV-induced apoptosis of human neutrophils. Although IL-16 processing occurs during apoptosis, IL-16C and MIF release was observed only during secondary necrosis of neutrophils. Screening a panel of microbial substances and proinflammatory cytokines did not identify a stimulus that induced the release of IL-16C and MIF independent of secondary necrosis. The data presented here suggest that IL-16 and MIF are neutrophil-derived inflammatory mediators released under conditions of insufficient clearance of apoptotic neutrophils, as typically occurs at sites of infection and autoimmunity.

Effect of hypertonic saline treatment on the inflammatory response after hydrochloric acid-induced lung injury in pigs

OBJECTIVES:Hypertonic saline has been proposed to modulate the inflammatory cascade in certain experimental conditions, including pulmonary inflammation caused by inhaled gastric contents. The present study aimed to assess the potential anti-inflammatory effects of administering a single intravenous dose of 7.5% hypertonic saline in an experimental model of acute lung injury induced by hydrochloric acid.METHODS:Thirty-two pigs were anesthetized and randomly allocated into the following four groups: Sham, which received anesthesia and were observed; HS, which received intravenous 7.5% hypertonic saline solution (4 ml/kg); acute lung injury, which were subjected to acute lung injury with intratracheal hydrochloric acid; and acute lung injury + hypertonic saline, which were subjected to acute lung injury with hydrochloric acid and treated with hypertonic saline. Hemodynamic and ventilatory parameters were recorded over four hours. Subsequently, bronchoalveolar lavage samples were collected at the end of the observation period to measure cytokine levels using an oxidative burst analysis, and lung tissue was collected for a histological analysis.RESULTS:Hydrochloric acid instillation caused marked changes in respiratory mechanics as well as blood gas and lung parenchyma parameters. Despite the absence of a significant difference between the acute lung injury and acute lung injury + hypertonic saline groups, the acute lung injury animals presented higher neutrophil and tumor necrosis factor alpha (TNF-α), interleukin (IL)-6 and IL-8 levels in the bronchoalveolar lavage analysis. The histopathological analysis revealed pulmonary edema, congestion and alveolar collapse in both groups; however, the differences between groups were not significant. Despite the lower cytokine and neutrophil levels observed in the acute lung injury + hypertonic saline group, significant differences were not observed among the treated and non-treated groups.CONCLUSIONS:Hypertonic saline infusion after intratracheal hydrochloric acid instillation does not have an effect on inflammatory biomarkers or respiratory gas exchange.

Cigarette smoke-induced damage-associated molecular pattern release from necrotic neutrophils triggers proinflammatory mediator release.

Cigarette smoking, the major causative factor for the development of chronic obstructive pulmonary disease, is associated with neutrophilic airway inflammation. Cigarette smoke (CS) exposure can induce a switch from apoptotic to necrotic cell death in airway epithelium. Therefore, we hypothesized that CS promotes neutrophil necrosis with subsequent release of damage-associated molecular patterns (DAMPs), including high mobility group box 1 (HMGB1), alarming the innate immune system. We studied the effect of smoking two cigarettes on sputum neutrophils in healthy individuals and of 5-day CS or air exposure on neutrophil counts, myeloperoxidase, and HMGB1 levels in bronchoalveolar lavage fluid of BALB/c mice. In human peripheral blood neutrophils, mitochondrial membrane potential, apoptosis/necrosis markers, caspase activity, and DAMP release were studied after CS exposure. Finally, we assessed the effect of neutrophil-derived supernatants on the release of chemoattractant CXCL8 in normal human bronchial epithelial cells. Cigarette smoking caused a significant decrease in sputum neutrophil numbers after 3 hours. In mice, neutrophil counts were significantly increased 16 hours after repeated CS exposure but reduced 2 hours after an additional exposure. In vitro, CS induced necrotic neutrophil cell death, as indicated by mitochondrial dysfunction, inhibition of apoptosis, and DAMP release. Supernatants from CS-treated neutrophils significantly increased the release of CXCL8 in normal human bronchial epithelial cells. Together, these observations show, for the first time, that CS exposure induces neutrophil necrosis, leading to DAMP release, which may amplify CS-induced airway inflammation by promoting airway epithelial proinflammatory responses.

Neutrophil Viability as a Clinical Outcome Marker in Mechanically Ventilated Critically Ill Trauma Patients: A Case Series

Abstract Background: Trauma in its early stages leads to an acute inflammatory condition affecting all cellular lines. Neutrophil granulocytes make up the largest population of human white blood cells and are fundamental to the innate immune system. The objective of our pilot study was to evaluate neutrophil death and viability alterations in critically ill trauma patients in correlation with their clinical outcome. Material and method: Critical ill trauma patients were enrolled in the study. In order to assess alterations in cellular death, blood samples were drawn using EDTA containing tubes and analyzed in the first twenty four hours after admission, then after forty eight and seventy two hours. Annexin V was used as a marker for apoptotic cells and propidium iodide for necrotic cells. Results: The first two cases exhibited an increase in cellular viability by the second day as shown by a small increase in neutrophil apoptosis and a decrease in neutrophil necrosis. These patients progressed to a positive clinical outcome. The second two cases showed slight modifications in either physiological or pathological cellular death, and increasing levels of cellular necrosis. These patients progressed to a negative clinical outcome. Conclusions: These cases suggest that neutrophil cell viability and death were associated with the patient’s clinical outcome.

Lung Mucosa Lining Fluid Modification of Mycobacterium tuberculosis to Reprogram Human Neutrophil Killing Mechanisms.

We have shown that human alveolar lining fluid (ALF) contains homeostatic hydrolases capable of altering the Mycobacterium tuberculosis cell wall and subsequently its interaction with human macrophages. Neutrophils are also an integral part of the host immune response to M. tuberculosis infection. Here we show that the human lung mucosa influences M. tuberculosis interaction with neutrophils, enhancing the intracellular killing of ALF-exposed M. tuberculosis and up-regulating the expression of tumor necrosis factor and interleukin 8. In contrast, ALF-exposed M. tuberculosis does not induce neutrophil apoptosis or necrosis, degranulation, or release of extracellular traps, and it decreases the oxidative response. These results suggest an important role for the human alveolar mucosa: increasing the innate capacity of the neutrophil to recognize and kill M. tuberculosis by favoring the use of intracellular mechanisms, while at the same time limiting neutrophil extracellular inflammatory responses to minimize their associated tissue damage.

Effect of mixed culture supernatants of Pseudomonas aeruginosa and Escherichia coli on apoptosis, necrosis, and oxidative activity of neutrophils.

Effect of mixed culture supernatants of Pseudomonas aeruginosa and Escherichia coli on apoptosis, necrosis, and oxidative activity of neutrophils.

Využití metod detekce apoptózy a nekrózy neutrofilních granulocytů krve skotu in vitro

The study was an in vitro analysis of bovine blood neutrophil apoptosis and necrosis based on the detection of their morphological and biochemical features. The experiment was carried out in six clinically healthy Holstein x Bohemian Red Pied crossbred virgin heifers aged 16 to 18 months. The fresh and in vitro cultivated blood were analysed by light and electron microscopy and flow cytometry. In fresh blood, non apoptotic or necrotic neutrophils were found by light microscopy. On the other hand, 3.34% apoptotic and 0.32% necrotic neutrophils were detected by flow cytometry. After four hours of incubation, 3.53% apoptotic and 1.37% necrotic neutrophils were found by light microscopy. In the same conditions, there were assessed 17.54% apoptotic and 0.58% necrotic neutrophils by flow cytometry. Correlation coefficient between light microscopy and flow cytometry in portion of apoptotic neutrophils was 0.625 (P&lt;0.05) and correlation coefficient between light microscopy and flow cytometry in portion of necrotic neutrophils was 0.983 (P&lt;0.05). There was found out that these detection methods (light microscopy and flow cytometry) are right combination for detection of apoptotic and necrotic bovine blood neutrophils.

S102 Lipoxin A4 Improves Efferocytosis Via Inhibition Of The Hmgb1 In Human Alveolar Macrophages

Introduction Effective clearance of apoptotic cells by macrophages, termed efferocytosis, is a pre-requisite for successful resolution of inflammation. High mobility group box protein 1 (HMGB1), is an alarmin that may promote inflammation as well as suppress phagocytosis. Lipoxin A 4 , represents one of a unique class of lipid mediators that possess a wide spectrum of anti-inflammatory and pro-resolution actions. We hypothesised that lipoxin A 4 may promote both apoptosis in neutrophils, and stimulate macrophage efferocytosis, acting as an antagonist to HMGB-1. Methods Neutrophils were obtained from healthy volunteers and cultured for 24 h with or without lipoxin A 4. Apoptosis of neutrophils was determined with Annexin V/SyTox staining by flow cytometry. HMGB-1 levels in Acute Respiratory Distress Syndrome (ARDS) bronchoalveolar lavage fluid (BALF) was measured by ELISA. The effects of HMGB-1 and lipoxin A 4 upon alveolar macrophage efferocytosis was assessed by measuring the ingestion of CMFDA labelled apoptotic neutrophils by flow cytometry. The PI3K (P85) protein expression was measured by western blotting. Results Treatment of lipoxin A4 (100 nM) increased the apoptosis of neutrophils (p = 0.0244), and reduced the dead (p = 0.0238) and necrotic neutrophils (p = 0.0358) compared to control (n = 8). BALF from patients with ARDS suppressed efferocytosis of apoptotic neutrophils. The effects of BALF correlated with HMGB-1 levels in the BALF fluid. HMGB-1 decreased efferocytosis (p Conclusions Lipxin A 4 in vitro promotes the apoptosis but not necrosis of neutrophils. In tandem it stimulates efferocytosis of alveolar macrophages. Elevated HMGB-1 in ARDS BALF suppresses efferocytosis. Lipoxin A 4 can block these effects of HMGB-1. The effect of lipoxin A4 increasing efferocytosis was through ALX–PI3K signalling pathways. Lipoxin A 4 may therefore have potential as a therapeutic agent to promote the resolution of neutrophilic inflammation in ARDS.

Toxic potencies of metabolite(s) of non-cylindrospermopsin producing Cylindrospermopsis raciborskii isolated from temperate zone in human white cells

Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria) has worldwide distribution and is well known for producing the toxic alkaloid, cylindrospermopsin (CYN). Strains unable to synthesize this compound but potentially toxic were recently identified in Europe. Here, for the first time the effect of cell-free extracts of a non-CYN-producing strain of C. raciborskii was studied in human cells (neutrophils and lymphocytes) isolated from healthy donors. The observed effects were compared to those induced by CYN (1.0–0.01μgmL−1). Short-term (1h) extract treatments resulted in altered viability of cells demonstrated by increased necrosis and apoptosis in neutrophils and elevated apoptosis in lymphocytes. CYN did not induce similar effects, regardless of the toxin concentration. Exposure of T-lymphocytes to 100% C. raciborskii extract in isolated and whole-blood 72h cultures resulted in decrease of proliferation by 20.6% and 32.5%, respectively. In comparison, exposure to 1.0μgmL−1 of CYN caused lymphocytes proliferation to be inhibited by 91.0% in isolated cultures and 56.5% in whole-blood assay. Significant antiproliferative properties were also found for 0.1μgmL−1 of CYN in whole-blood culture. From the results we conclude that strains occurring in temperate zones may pose a threat to human health through the production of hitherto unknown metabolites that reveal a toxic pattern different to that of CYN. At the same time our study demonstrates that CYN is a powerful but slowly-acting toxin in human immune cells.