Neutrophil Degranulation Responses Research Articles

Deciphering lipoprotein(a) atherogenicity with use of high-throughput proteomics

Abstract Background Accumulating observational and genetic evidence suggests that lipoprotein(a) [Lp(a)], is significantly more atherogenic than other apolipoprotein-B containing particles. Systemic inflammation is thought to mediate the atherogenic effects of Lp(a). Previous studies have predominantly focused on single inflammatory biomarkers, an approach that fails to capture the broader molecular response to elevated Lp(a) levels. Purpose The aim of this study was to derive a proteome-wide signature of plasma Lp(a) concentrations and subsequently identify the pathways through which Lp(a) influences the risk of coronary artery disease (CAD). Methods This study included 41,123 UK Biobank participants with baseline Lp(a) measurements who also underwent a proteomic assessment. Relative concentration of 2,920 proteins was quantified in EDTA plasma samples using a high-throughput proximity extension assay. Lp(a)-associated proteins were identified in linear regression models, adjusting for age, sex, body mass index, race, smoking status, estimated glomerular filtration rate, medication use, and apolipoprotein B. Proteins linked with Lp(a) concentration were then evaluated for their association with incident CAD risk using Cox proportional hazards models. Significant traits were further included in pathway enrichment analysis. Results Out of 2,920 analyzed proteins (Figure 1), 140 showed a positive association with Lp(a) concentration, while 336 exhibited a negative association after adjustment for multiple testing (False discovery rate < 0.05). The strongest positive associations were observed for apolipoprotein F, group 10 secretory phospholipase A, and C4b-binding protein beta chain. The strongest negative associations were observed for asporin, aminoacylase-1, and retinol-binding protein 5. Among 140 proteins positively associated with Lp(a), 49 were associated with an increased risk of CAD. Conversely, among the 336 proteins negatively associated with Lp(a), 10 were associated with a decreased risk of CAD. Pathway enrichment analysis (Figure 2) indicated that proteins linked with both Lp(a) levels and CAD risk predominantly upregulated pathways related to peptide-ligand receptor signalling, platelet degranulation, neutrophil degranulation, and cytosolic calcium response. Conversely, pathways related to HDL remodeling, PI3K/AKT, KIT, and nuclear receptor signalling were downregulated. Conclusions The greater atherogenic potential of Lp(a) may be attributable to the modulation of several molecular pathways, particularly those involving platelet and neutrophil responses, cell signalling, and lipoprotein metabolism.

Impact of Hypoxia on Neutrophil Degranulation and Inflammatory Response in Alpha-1 Antitrypsin Deficiency Patients.

Alpha-1 antitrypsin deficiency (AATD) is an inflammatory disorder where neutrophils play a key role. Excessive neutrophil activation leads to local hypoxia and tissue damage. Most research on neutrophil function has been conducted under atmospheric conditions (21% O2), which may not represent physiological or pathological conditions. This study aimed to determine the effects of hypoxia on neutrophil degranulation and cytokine production in AATD patients. Neutrophils isolated from 54 AATD patients (31 MZ; 8 SZ; 15 ZZ) and 7 controls (MM) were exposed to hypoxia (1% O2) for 4 h. Neutrophil degranulation was assessed by measuring elastase (NE), myeloperoxidase (MPO), lactoferrin, and matrix metalloproteinase-9 (MMP-9) levels using immunoassay-based methods. Pro-inflammatory (IL-8, IL-1 beta, IL-6, and TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokine levels were assessed by a Luminex-based method. Our results indicate a significantly increased release of NE (p = 0.015), MPO (p = 0.042), lactoferrin (p = 0.015), and MMP-9 (p = 0.001) compared to controls. Pro-inflammatory cytokines show a significant rise in IL-8 (p = 0.019), a trend towards increased IL-1 beta (p = 0.3196), no change in IL-6 (p = 0.7329), and reduced TNF-alpha (p = 0.006). Anti-inflammatory cytokines show increased IL-4 (p = 0.057) and decreased IL-10 (p = 0.05703). Increased neutrophil degranulation and inflammatory phenotype are observed in AATD neutrophils under physiological hypoxia.

Vaccinia virus Tiantan strain blocks host antiviral innate immunity and programmed cell death by disrupting gene expression

The vaccinia virus Tiantan (VTT) is widely utilized as a smallpox vaccine in China and holds significant importance in the prevention of diseases stemming from poxvirus infections. Nevertheless, few studies have investigated the influence of VTT infection on host gene expression. In this study, we constructed time series transcriptomic profiles of HeLa cells infected with both VTT and western reserve (WR) strains. We observed similar patterns of viral gene expression, while the expression levels of host genes varied between the two strains. There was an immediate and significant repression of host gene expression, particularly in genes associated with oxidative phosphorylation. Conversely, genes involved in nerve growth factor (NGF)-stimulated transcription were significantly activated. The upregulation of genes linked to the ribonucleic acid (RNA)-induced silencing complex (RISC) suggested a potential role for posttranscriptional regulation in the interaction between the vaccinia virus and the host. In the later stages of infection, pathways such as extracellular matrix organization, neutrophil degranulation, complement and interferon responses, translation, and programmed cell death are largely inhibited. A significant number of host genes exhibit correlations with changes in the expression levels of viral genes. The host genes that are negatively correlated with viral genes are mainly enriched in pathways associated with translation and the response to viral infection. This study significantly contributes to advancing our understanding of the dynamics between the vaccinia virus and the host, improving the application of VTTs and facilitating the development of effective vaccines against diseases such as smallpox and monkeypox.

Identifying Key Genes and Their Associated Molecular Pathways in Lupus Nephritis-Osteoporosis: An In-Silico Analysis

Nephritis and osteoporosis are debilitating medical conditions that significantly impact human health and reduce quality of life. To develop potential therapeutic strategies for these disorders necessitates understanding the genetic and molecular mechanisms. Here, we employed bioinformatics techniques purposed to find key genes and associated pathways responsible for nephritis-osteoporosis comorbidity. Six microarray datasets of systemic lupus erythematosus (SLE) and osteoporosis were retrieved from the Gene Expression Omnibus (GEO) database. Post normalization of data sets LIMMA package was utilized for differential expression analysis, among the datasets 44 differentially expressed genes (DEGs) were identified. The identified 44 genes were further analyzed for gene ontology (GO) where it was found that these genes are involved in defense response, organism interactions, and response to external stimuli. In predicting the molecular function, they were involved in several biological processes including binding to lipopolysaccharides and having peptidase and hydrolase activities. Firstly, the identified genes were primarily associated with certain granules such as specific granules and secretory granules in the aspect of cellular components. Enrichment analysis pointed out the potential pathways linked to the immune system, neutrophil degranulation, innate immunity, and immune response to tuberculosis. To examine interactions among DEGs, a complex protein-protein interaction (PPI) network was built, resulting in the identification of seven hub genes, CXCL8, ELANE, LCN2, MMP8, IFIT1, MX1, and ISG15. The study suggests that these elucidated hub genes might have high potential to be exploited as promising biomarkers and therapeutic targets in nephritis-osteoporosis. Taken together, this study provided deeper insights into the genetic and molecular basis for the comorbidity of nephritis and osteoporosis.

Red cell exchange modulates neutrophil degranulation responses in sickle cell disease.

Neutrophils in sickle cell disease (SCD) are activated, contributing to disease. Red cell exchange (RCE), with the goal of lowering hemoglobin S (HbS), is an important part of therapy for many SCD patients. Whether RCE impacts neutrophil reactivity is unknown. To determine the effect of RCE on neutrophil activation, SCD patients undergoing RCE in steady-state were enrolled. Neutrophil degranulation responses were examined before/after RCE. Kinetic studies were completed to determine the duration of the effect of RCE on neutrophil function. Degranulation results were examined in relation to white blood cell count, neutrophil count, and HbS levels. The effect of RCE on RBC phosphatidylserine (PS) exposure was examined as a possible contributor to modulation of neutrophil function by RCE. Twenty-two patients with SCD, genotype SS, who underwent RCE (average pre-RCE HbS 33 ± 14%) were included for the study. RCE significantly decreased neutrophil degranulation responses. The effect of RCE on neutrophil activation was unrelated to cell count and instead directly correlated with HbS. The effect of RCE on neutrophil activation was sustained over several days post-apheresis. Furthermore, while increased RBC PS exposure results in increased neutrophil degranulation, RCE decreases RBC PS exposure. To our knowledge, this is the first study demonstrating that RCE significantly decreases neutrophil activation in a sustained HbS-dependent manner. Modulation of PS exposure by RCE may be a contributing mechanism by which RCE modulates neutrophil activation. These studies raise the possibility that modulation of neutrophil activation contributes significantly to the therapeutic effect of RCE.

Expression of Concern: Pyk2 Is Required for Neutrophil Degranulation and Host Defense Responses to Bacterial Infection.

Expression of Concern: Pyk2 Is Required for Neutrophil Degranulation and Host Defense Responses to Bacterial Infection.

The genomic landscape of ANCA-associated vasculitis: Distinct transcriptional signatures, molecular endotypes and comparison with systemic lupus erythematosus.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) present with a complex phenotype and are associated with high mortality and multi-organ involvement. We sought to define the transcriptional landscape and molecular endotypes of AAVs and compare it to systemic lupus erythematosus (SLE). We performed whole blood mRNA sequencing from 30 patients with AAV (granulomatosis with polyangiitis/GPA and microscopic polyangiitis/MPA) combined with functional enrichment and network analysis for aberrant pathways. Key genes and pathways were validated in an independent cohort of 18 AAV patients. Co-expression network and hierarchical clustering analysis, identified molecular endotypes. Multi-level transcriptional overlap analysis to SLE was based on our published data from 142 patients. We report here that "Pan-vasculitis" signature contained 1,982 differentially expressed genes, enriched in leukocyte differentiation, cytokine signaling, type I and type II IFN signaling and aberrant B-T cell immunity. Active disease was characterized by signatures linked to cell cycle checkpoints and metabolism pathways, whereas ANCA-positive patients exhibited a humoral immunity transcriptional fingerprint. Differential expression analysis of GPA and MPA yielded an IFN-g pathway (in addition to a type I IFN) in the former and aberrant expression of genes related to autophagy and mRNA splicing in the latter. Unsupervised molecular taxonomy analysis revealed four endotypes with neutrophil degranulation, aberrant metabolism and B-cell responses as potential mechanistic drivers. Transcriptional perturbations and molecular heterogeneity were more pronounced in SLE. Molecular analysis and data-driven clustering of AAV uncovered distinct transcriptional pathways that could be exploited for targeted therapy. We conclude that transcriptomic analysis of AAV reveals distinct endotypes and molecular pathways that could be targeted for therapy. The AAV transcriptome is more homogenous and less fragmented compared to the SLE which may account for its superior rates of response to therapy.

Neonatal prophylactic antibiotics after preterm birth affect plasma proteome and immune development in pigs.

Most preterm infants receive antibiotics to prevent serious infections shortly after birth. However, prolonged antibiotic treatment predisposes to gut dysbiosis and late-onset sepsis. Using preterm pigs as model, we hypothesized that neonatal prophylactic antibiotics impair systemic immune development beyond the days of antibiotic treatment. Preterm pigs (90% gestation) were fed formula for 9 days, treated with sterile water (CON) or enteral antibiotics from day 1 to 4. On days 5 and 9, blood was collected for haematology, in vitro LPS stimulation, and plasma proteomics. Antibiotic treatment altered the abundance of 21 and 47 plasma proteins on days 5 and 9, representing 6.6% and 14.8% of the total annotated proteins, respectively. Most antibiotics-induced proteome changes related to complement cascade, neutrophil degranulation, and acute phase responses. Neutrophil and lymphocyte counts were higher in antibiotics-treated pigs on day 5 but did not change from days 5-9, in contrast to increasing cell counts in CON. The antibiotics treatment suppressed TNF-alpha and IL-10 responses to in vitro LPS challenge on day 5, 7 and 9. Few days of antibiotics treatment following preterm birth alter the plasma proteome and inhibit systemic immune development, even beyond the days of treatment. 1. Neonatal prophylactic antibiotics alter the plasma proteome and suppress systemic immune development in preterm pigs 2. The effects of prophylactic antibiotics last beyond the days of treatment. 3. Neonatal antibiotics treatment for compromised human newborns may predispose to longer-term risks of impaired immunity and infections.

Longitudinal proteomic investigation of COVID-19 vaccination.

Although the development of COVID-19 vaccines has been a remarkable success, the heterogeneous individual antibody generation and decline over time are unknown and still hard to predict. In this study, blood samples were collected from 163 participants who next received two doses of an inactivated COVID-19 vaccine (CoronaVac®) at a 28-day interval. Using TMT-based proteomics, we identified 1,715 serum and 7,342 peripheral blood mononuclear cells (PBMCs) proteins. We proposed two sets of potential biomarkers (seven from serum, five from PBMCs) at baseline using machine learning, and predicted the individual seropositivity 57 days after vaccination (AUC = 0.87). Based on the four PBMC's potential biomarkers, we predicted the antibody persistence until 180 days after vaccination (AUC = 0.79). Our data highlighted characteristic hematological host responses, including altered lymphocyte migration regulation, neutrophil degranulation, and humoral immune response. This study proposed potential blood-derived protein biomarkers before vaccination for predicting heterogeneous antibody generation and decline after COVID-19 vaccination, shedding light on immunization mechanisms and individual booster shot planning.

Blood plasma proteome alteration after local tissue damage induced by Bothrops erythromelas snake venom in mice

Snakes of the genus Bothrops are responsible the most snakebites in the Brazil, causing a diverse and complex pathophysiological condition. Bothrops erythromelas is the main specie of medical relevance found in the Caatinga from the Brazilian Northeast region. The pathophysiological effects involving B. erythromelas snakebite as well as the organism reaction in response to this envenomation are not so explored. Thus, edema was induced in mice paws using 2.5 μg or 5.0 μg of B. erythromelas venom, and the percentage of edema was measured. Plasma was collected 30 minutes after the envenomation-induced in mice and analyzed by mass spectrometry. It was identified a total of 112 common plasma proteins differentially abundant among experimental groups, which are involved with the complement system and coagulation cascades, oxidative stress, neutrophil degranulation, platelets degranulation and inflammatory response. Apolipoprotein A1 (Apoa), serum amyloid protein A-4 (Saa4), adiponectin (Adipoq) showed up-regulated in mice plasma after injection of venom, while fibulin (Fbln1), factor XII (F12) and vitamin K-dependent protein Z (Proz) showed down-regulated. The results indicate a protein pattern of thrombo-inflammation to the B. erythromelas snakebite, evidencing potential biomarkers for monitoring this snakebite, new therapeutic targets and its correlations with the degree of envenomation once showed modulations in the abundance among the different groups according to the amount of venom injected into the mice.

An experimental in silico study on COVID-19: Response of neutrophil-related genes to antibiotics.

Background and AimsAll components of the immune system are involved in alleviating severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. Further research is required to provide detailed insights into COVID‐19‐related immune compartments and pathways. In addition, a significant percentage of hospitalized COVID‐19 patients suspect bacterial infections and antimicrobial resistance occurs following antibiotics treatment. The aim of this study was to evaluate the possible effects of antibiotics on the response of neutrophil‐related genes in SARS‐CoV‐2 patients by an experimental in silico study.MethodsThe two data sets GSE1739 and GSE21802 including 10 SARS positive patients and 35 influenza A (H1N1) patients were analyzed, respectively. Differentially expressed genes (DEGs) between these two data sets were determined by GEO2R analysis and the Venn diagram online tool. After determining the hub genes involved in immune responses, the expression of these genes in 30 COVID‐19 patients and 30 healthy individuals was analyzed by real‐time polymerase chain reaction (PCR). All patients received antibiotics, including levofloxacin, colistin, meropenem, and ceftazidime.ResultsGEO2R analysis detected 240 and 120 DEGs in GSE21802 and GSE1739, respectively. Twenty DEGs were considered as enriched hub genes involved in immune processes such as neutrophil degranulation, neutrophil activation, and antimicrobial humoral response. The central nodes were attributed to the genes of neutrophil elastase (ELANE), arginase 1 (ARG‐1), lipocalin 2 (LCN2), and defensin 4 (DEFA4). Compared to the healthy subjects, the expression of LCN2 and DEFA4 were significantly reduced in COVID‐19 patients. However, no significant differences were observed in the ELANE and AGR‐1 levels between COVID‐19 subjects and the control group.ConclusionsActivation and degranulation of neutrophils were observed mainly in SARS, and H1N1 infection processes and antibiotics administration could affect neutrophil activity during viral infection. It can be suggested that antibiotics can decrease inflammation by restoring the expression of neutrophil‐related genes in COVID‐19 patients.

Gene Differential Expression and Interaction Networks Illustrate the Biomarkers and Molecular Mechanisms of Atherosclerotic Cerebral Infarction.

Atherosclerotic cerebral infarction (ACI) seriously threatens the health of the senile patients, and the strategies are urgent for the diagnosis and treatment of ACI. This study investigated the mRNA profiling of the patients with ischemic stroke and atherosclerosis via excavating the datasets in the GEO database and attempted to reveal the biomarkers and molecular mechanism of ACI. In this study, GES16561 and GES100927 were obtained from Gene Expression Omnibus (GEO) database, and the related differentially expressed genes (DEGs) were analyzed with R language. Furthermore, the DEGs were analyzed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Besides, the protein-protein interaction (PPI) network of DEGs was analyzed by STRING database and Cytoscape. The results showed that 133 downregulated DEGs and 234 upregulated DEGs were found in GES16561, 25 downregulated DEGs and 104 upregulated DEGs were found in GSE100927, and 6 common genes were found in GES16561 and GES100927. GO enrichment analysis showed that the functional models of the common genes were involved in neutrophil activation, neutrophil degranulation, neutrophil activation, and immune response. KEGG enrichment analysis showed that the DEGs in both GSE100927 and GSE16561 were connected with the pathways including Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Phagosome, Antigen processing and presentation, and Staphylococcus aureus infection. The PPI network analysis showed that 9 common DEGs were found in GSE100927 and GSE16561, and a cluster with 6 nodes and 12 edges was also identified by PPI network analysis. In conclusion, this study suggested that FCGR3A and MAPK pathways were connected with ACI.

Variation in Neutrophil Function Among Healthy Subjects Influences Response to Bacteria

Introduction: We have recently shown that healthy subjects have variable neutrophil degranulation responses to antigen/antibody immune complexes (ICs; Duarte ME Blood 2019). With repeated testing over 2 years, we have shown that susceptibility to neutrophil activation represents a fixed phenotype: some healthy subjects have neutrophils which robustly degranulate in response to ICs, while others have neutrophils that are minimally reactive (>30-fold variation in release of MMP9, representative of tertiary granules). Whether this variation in degranulation responses is specific to ICs or is applicable to other stimulants is not known. Because neutrophils are involved in the first steps of host defense against bacterial pathogens, we aimed to determine if these phenotypes are associated with differences in neutrophil effector functions which may contribute to host defense. Methods/Results: To determine if observed neutrophil phenotypes are relevant to host defense, we compared neutrophil effector responses from "high" and "low" donors after being challenged with bacterial products including the bacterial tripeptide N-formyl-met-leu-phe (fMLP), LPS, and Staphylococcus aureus (USA300) supernatant. As a model IC, we chose to use KKO-platelet factor 4/heparin ICs, as neutrophil responses to this IC have been previously characterized by our lab. As a first step, we examined several healthy subjects and compared degranulation responses to ICs, fMLP, and LPS to determine if susceptibility to degranulation was preserved. As shown in Figure 1, the quantity of MMP9 released in response to ICs was highly correlated with MMP9 released in response to fMLP (Fig 1A) and to LPS (Fig 1B, r = 0.9 for both). We then determined if neutrophil phenotypes were preserved in response to bacteria. As shown in Figure 2, neutrophils from "high" and "low" donors differentially degranulated in response to Staphylococcus aureus supernatant, with continued preservation of the neutrophil phenotype. We next aimed to determine if the neutrophil phenotype was associated with differences in other neutrophil effector functions, besides degranulation, which are relevant to pathogen defense. First, we focused on neutrophil extracellular trap (NET) release, which has an important role in host defense. As shown in Figure 3, "high" and "low" donors differed in NET release in response to ICs, as quantified by visualization using immunofluorescent microscopy. This finding is supported by our previous report that neutrophil phenotypes are correlated with differences in MPO release, a NET component (Duarte ME, et al 2019). We also examined reactive oxygen species (ROS) generation, a primary mechanism by which neutrophils eliminate pathogens. As shown in Figure 4, using DCFH-DA, a fluorescent probe, we show that "high" and "low" donors differ in ROS generation in response to PMA and fMLP. Conclusions: Taken together, our studies demonstrate that neutrophil responses to a variety of stimuli (ICs, LPS, and fMLP) are highly concordant among healthy donors with "high" or "low" neutrophil phenotypes and that these differences in neutrophil effector functions are likely relevant to host defense. Further studies are needed to determine how differences in innate neutrophil function may affect clinical outcomes during bacterial infection. Disclosures Arepally: Apotex: Consultancy, Research Funding; Biokit: Consultancy, Patents & Royalties; Veralox Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Annexon Biosciences: Consultancy, Other; Alexion: Other; Novartis: Consultancy.

Disruption of hepatic small heterodimer partner induces dissociation of steatosis and inflammation in experimental nonalcoholic steatohepatitis

Disruption of hepatic small heterodimer partner induces dissociation of steatosis and inflammation in experimental nonalcoholic steatohepatitis

Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection.

Neutrophils are theprimary immune cell recruited to the site of bacterial infection, where they can rapidly deploy vesicles filled with various pro-inflammatory and anti-microbial proteins. This degranulation process, combined with oxidative and nitrosative mechanisms, is a major part of the initial host response to kill microorganisms. Neutrophils are one of the main cell types that interact with Yersinia pestis during infection, which is often lethal in the absence of prompt antibiotic treatment. Intradermal inoculation of Y. pestis results in bubonic plague, and inhalation of aerosolized droplets containing Y. pestis results in pneumonic plague. Although neutrophils are recruited to the site of inoculation during both bubonic and pneumonic plague, the neutrophils fail to clear Y. pestis, and, during pneumonic plague, contribute to the development of severe pneumonia. Subverting neutrophil responses is critical to the development of fulminant disease, yet the mechanisms by which Y. pestis impairs neutrophils are poorly understood. Cell culture models are important tools for studying Y. pestis interactions with immune cells. We describe a cell culture model for the infection of human neutrophils with Y. pestis. Neutrophils are isolated from human peripheral blood at high purity and subsequently infected with Y. pestis. We specifically focus on the application of this in vitro infection assay to the analysis of neutrophil degranulation responses.

The BLT1 Inhibitory Function of α-1 Antitrypsin Augmentation Therapy Disrupts Leukotriene B4 Neutrophil Signaling.

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases.

Evidence for Anti-Inflammatory Effects of Exercise in CKD

CKD is associated with a complex state of immune dysfunction characterized by immune depression, predisposing patients to infections, and immune activation, resulting in inflammation that associates with higher risk of cardiovascular disease. Physical exercise may enhance immune function and exert anti-inflammatory effects, but such effects are unclear in CKD. We investigated the separate effects of acute and regular moderate-intensity aerobic exercise on neutrophil degranulation (elastase release), activation of T lymphocytes (CD69 expression) and monocytes (CD86 and HLA-DR expression), and plasma inflammatory markers (IL-6, IL-10, soluble TNF-receptors, and C-reactive protein) in patients with predialysis CKD. A single 30-minute (acute) bout of walking induced a normal pattern of leukocyte mobilization and had no effect on T-lymphocyte and monocyte activation but improved neutrophil responsiveness to a bacterial challenge in the postexercise period. Furthermore, acute exercise induced a systemic anti-inflammatory environment, evidenced by a marked increase in plasma IL-10 levels (peaked at 1 hour postexercise), that was most likely mediated by increased plasma IL-6 levels (peaked immediately postexercise). Six months of regular walking exercise (30 min/d for 5 times/wk) exerted anti-inflammatory effects (reduction in the ratio of plasma IL-6 to IL-10 levels) and a downregulation of T-lymphocyte and monocyte activation, but it had no effect on circulating immune cell numbers or neutrophil degranulation responses. Renal function, proteinuria, and BP were also unaffected. These findings provide compelling evidence that walking exercise is safe with regard to immune and inflammatory responses and has the potential to be an effective anti-inflammatory therapy in predialysis CKD.

Inhibition of neutrophil respiratory burst and degranulation responses by CVT-E002, the main active ingredient in COLD-FX

Background Human peripheral blood neutrophils contribute to the first line of defence in the immune system and are critical for maintaining health and immunity against opportunistic infections. Neutrophils and their granule-derived mediators are frequently found elevated in patient samples in viral infections, asthma exacerbations, and other respiratory ailments. COLD-FX has been shown to reduce the symptoms and severity of respiratory tract viral infections. Our hypothesis is that COLD-FX modulates neutrophil activity. To determine the effects of COLD-FX on neutrophils, peripheral blood neutrophils (>97% purity) were isolated from healthy human volunteers. Methods Neutrophils were preincubated with varying doses of CVT-E002 (0.01-1 mg/ml), the active ingredient of COLD-FX, for 30, 60, and 120 min. Extracellular ROS production was measured by cytochrome c reduction from neutrophils stimulated with 50 ng/ml phorbol myristate acetate for up to 60 min. Degranulation was measured by the presence of extracellular myeloperoxidase, a marker of the azurophilic granules, in neutrophils stimulated with cytochalasin B and f-Met-Leu-Phe for 15 min.

Pyk2 is required for neutrophil degranulation and host defense responses to bacterial infection.

The appropriate regulation of neutrophil activation is critical for maintaining host defense and limiting inflammation. Polymorphonuclear neutrophils (PMNs) express a number of cytoplasmic tyrosine kinases that regulate signaling pathways leading to activation. One of the most highly expressed, but least studied, kinases in PMNs is proline rich kinase 2 (Pyk2). By analogy to the related focal adhesion kinase, Pyk2 has been implicated in regulating PMN adhesion and migration; however, its physiologic function has yet to be described. Using pyk2(-/-) mice, we found that this kinase was required for integrin-mediated degranulation responses, but was not involved in adhesion-induced cell spreading or activation of superoxide production. Pyk2-deficient PMNs also manifested reduced migration on fibrinogen-coated surfaces. The absence of Pyk2 resulted in a severe reduction in paxillin and Vav phosphorylation following integrin ligation, which likely accounts for the poor degranulation and cell migration. Pyk2(-/-) mice were unable to efficiently clear infection with Staphylococcus aureus in a skin abscess model, owing in part to the poor release of granule contents at the site of infection. However, Pyk2-deficient PMNs responded normally to soluble agonists, demonstrating that this kinase functions mainly in the integrin pathway. These data demonstrate the unrealized physiologic role of this kinase in regulating the adhesion-mediated release of PMN granule contents.

No effect of a 30-h period of sleep deprivation on leukocyte trafficking, neutrophil degranulation and saliva IgA responses to exercise

A one night period without sleep is not uncommon amongst athletes travelling across time zones and military personnel during training and operations. However, the effect of one night without sleep on immune indices in response to strenuous exercise remains unknown. The objective was to determine the effect of one night without sleep on immune indices in response to subsequent strenuous exercise. Using a repeated measures cross-over design, on one occasion eleven male participants slept normally (CON) and on another they were sleep deprived for 30 h (SDEP). After 30 h participants performed 30 min steady state (SS) treadmill exercise at 60% VO2max followed by a 30 min treadmill time trial (TT). Blood and saliva samples were collected at 0 h, 30 h, post-SS, post-TT, 2 h post-TT and 18 h post-TT. Circulating leukocyte and T-lymphocyte subset counts, bacterially-stimulated neutrophil degranulation, saliva secretory immunoglobulin A (S-IgA) and plasma cortisol were determined. No trial x time interactions were observed for immune indices and plasma cortisol. A leukocytosis, neutrophilia, and lymphocytosis was observed post-TT compared with 30 h (P < 0.01). Also, at post-TT compared with 30 h an increase in circulating T-lymphocyte CD3 + (55%) and CD8 + (67%) counts (P < 0.05), a decrease in neutrophil degranulation (20%; P < 0.05) and an increase in S-IgA concentration (83%) was observed (P < 0.01). Plasma cortisol concentration increased post-TT (62%) compared with post-SS (P < 0.01). In conclusion, a 30 h period of sleep deprivation does not alter leukocyte trafficking, neutrophil degranulation or S-IgA responses either at rest or after submaximal and strenuous exercise.