Polymorphonuclear Neutrophil Granulocytes Research Articles

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Short chain fatty acids reduce the respiratory burst of human neutrophils in response to cystic fibrosis isolates of Staphylococcus aureus

Short chain fatty acids (SCFA) are produced by anaerobic bacteria. The most common SCFAs are acetate, propionate and butyrate. SCFAs have been implicated in several inflammatory diseases including cystic fibrosis (CF) where they are present in the airways at millimolar concentrations. Staphylococcus aureus is one of the main respiratory pathogens in CF. Polymorphonuclear neutrophil granulocytes (PMN) represent the most important immune defense the host uses against S. aureus. However, the reason why PMNs are unable to clear S. aureus in CF remains largely unclear. We hypothesized that SCFAs impair effector functions of PMNs in response to S. aureus. To test this, human PMNs were exposed to CF clinical isolates of S. aureus in vitro in the presence or absence of SCFAs and effector functions of PMNs were assessed. Our data show that SCFAs do not affect the viability of PMNs and do not stimulate the release of neutrophil extracellular traps (NET) from human PMNs. Production of reactive oxygen species (ROS), another important antimicrobial function of PMNs, on the other hand, was significantly inhibited by SCFAs in response to the bacterium. SCFAs did not compromise the ability of PMNs to kill CF isolates of S. aureus in vitro. Overall, our results provide new knowledge into the interactions between SCFAs and the immune system, and indicate that SCFAs produced by anaerobic bacteria in the CF lung could interfere with reactive oxidant production of PMNs in response to S. aureus, one of the prominent respiratory pathogens in this disease.

Myeloperoxidase-Sensitive T1 and T2 Switchable MR Imaging for Diagnosis of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is the critical stage in the development of nonalcoholic fatty liver disease (NAFLD) from simple and reversible steatosis to irreversible cirrhosis and even hepatocellular carcinoma (HCC). Thus, the diagnosis of NASH is important for preventing the progress of NAFLD into a fatal condition. The oxidative enzyme myeloperoxidase (MPO), which is mostly produced by polymorphonuclear neutrophil granulocytes (NEU), has been identified as a key player in lipid peroxidation in inflamed tissues. Considering that the expression of MPO was much higher in NASH than in the nonalcoholic fatty liver (NAFL) with steatosis, we designed a nanoparticle platform based on ultrasmall iron oxide (USIO) nanoparticles to realize MPO-sensitive NASH diagnosis. After modification of USIO nanoparticles with amphiphilic poly(ethylene glycol) (PEG) and conjugation with 5-hydroxytryptamine (5HT), a physiological substrate for MPO, the final nanocomposite (USIO-DA-PEG-5HT) revealed MPO-mediated aggregation at the inflammatory site of NASH. Meanwhile, the intrinsic T1-weighted magnetic resonance (MR) signal of dispersed USIO-DA-PEG-5HT nanoparticles diminishes, while the T2-weighted MR signal is amplified owing to the aggregation effect. These USIO-DA-PEG-5HT nanoprobes offer great potential for improving NASH MR imaging diagnostic accuracy and sensitivity compared to existing molecular MR contrast agents with a single imaging modality.

Is the IL1RA/IL1B Ratio a Suitable Biomarker for Subclinical Endometritis in Dairy Cows?

The adequate expression of cytokines is essential for the prevention and healing of bovine endometrial inflammation. This study investigated the intra-uterine concentration of the proinflammatory cytokine interleukin (IL)1B and its antagonist IL1RA in cows with and without subclinical endometritis (SE). Samples were taken from 37 uteri at the abattoir and 26 uteri in vivo. Uterine secretion samples were classified as showing no signs of SE (SEneg; polymorphonuclear neutrophil granulocyte (PMN) < 5%) or showing signs of SE (SEpos; PMN ≥ 5%). Concentrations and ratios for IL1B and IL1RA were measured using a commercial and a newly established AlphaLISA kit, respectively. In both groups, a higher concentration of IL1B was detected in the SEpos group compared with the SEneg group (abattoir: p = 0.027; in vivo p < 0.001). No significant differences were observed in the concentration of IL1RA (p > 0.05). In uterine secretion samples retrieved in vivo, a lower IL1RA/IL1B ratio was detected in the SEpos group compared with the SEneg group (p = 0.002). The results of this study highlight the important role of IL1B and IL1RA during endometritis and the potential of the IL1RA/IL1B ratio as a possible biomarker for SE.

Murine burn lesion model for studying acute and chronic wound infections.

Acute wounds, such as thermal injury, and chronic wounds are challenging for patients and the healthcare system around the world. Thermal injury of considerable size induces immunosuppression, which renders the patient susceptible to wound infections, but also in other foci like the airways and urinary tract. Infected thermal lesions can progress to chronic wounds with biofilm making them more difficult to treat. While animal models have their limitations, murine wound models are still the best tool at the moment to identify strategies to overcome these challenges. Here, we present a murine burn model, which has been developed to study biofilm formation, the significance of wound healing, and for identifying novel treatment candidates. Investigating the effect of a thermal injury in mice, we observed that 48 h after introduction of the injury, the mice showed a reduction in polymorphonuclear neutrophil granulocytes (PMNs) and a reduced capacity for phagocytosis and oxidative burst. Regarding the chronic wound, Pseudomonas aeruginosa biofilm arrested wound healing and kept the wound in an inflammatory state, but suppressing PMN function by means of the PMN factor S100A8/A9, corresponding to observations in human venous leg ulcers. Monotherapy and dual treatment with S100A8/A9 and ciprofloxacin on P. aeruginosa biofilm-infected murine wounds have been investigated. In combination, S100A8/A9 and ciprofloxacin reduced the bacterial quantity, lowered the proinflammatory response, and increased anti-inflammatory cytokines after 4 days of treatment. When the treatment was prolonged, an additional prevention of resistance development was detected in all the dual-treated mice. In the present review, we provide data on using the murine model for research with the aim of better understanding pathophysiology of wounds and for identifying novel treatments for humans suffering from these lesions.

Investigation of anti-neutrophil cytoplasmic antibody presence with indirect immunofluorescence and enzyme-linked immunosorbent assay

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies that react with proteins expressed mainly in cytoplasmic granules of polymorphonuclear neutrophil granulocytes (PMNs). The ANCA test is used to diagnose small vessel vasculitis and to monitor inflammatory activity. ANCA was initially detected using indirect immunofluorescence (IIF), which allowed differentiation of different patterns, such as p-ANCA (perinuclear) and c-ANCA (cytoplasmic). It is now common to detect antibodies by immunochemical assays using purified proteins, such as Enzyme-Linked Immunosorbent Assay (ELISA). In our study, we evaluated ANCA test results studied with IIF and ELISA methods and recorded patient diagnoses in the system. Serum samples of 4524 patients who were thought to have autoimmunity in their etiology were evaluated for ANCA presence. In accordance with the recommendation of the manufacturer (Euroimmun AG, Lübeck, Germany), serum IFA technique was evaluated for the presence of p-ANCA, c-ANCA. ELISA test (Alegria, Orgentec) was used to detect antibodies against MPO and PR3. The number of ANCA IIF positive patients was 525(11.6%). When we look at the distribution of ANCAs, 275(52.5%) formalin sensitive pANCA, 95 (18%) formalin resistant pANCA, 60 formalin sensitive cANCA(11.5%), 95(18%) formalin resistant cANCA. 18 (3.4%) of ANCA IIFA positives had PR3 antigen and 22 (4.1%) had significant antibody elevation against MPO antigen., ANCA IIF positive samples, according to the diagnosis of information registered in the operating system, consist of 424 (80.8%) autoimmune and inflammatory diseases according to disease groups, 36 (6.9%) malignancies, 18 (3.4%) infectious diseases, 47 (8.9%) are other diseases which are not included in these groups. ANCA is a determinant for many diseases, especially vasculitis. ANCA, which we found in a large number of different disease groups, was found to be an indicator that should be used in the diagnosis and follow-up of many autoimmune and inflammatory diseases.

Enhancement of epidermal growth factor receptor antibody tumor immunotherapy by glutaminyl cyclase inhibition to interfere with CD47/signal regulatory protein alpha interactions.

Integrin associated protein (CD47) is an important target in immunotherapy, as it is expressed as a “don't eat me” signal on many tumor cells. Interference with its counter molecule signal regulatory protein alpha (SIRPα), expressed on myeloid cells, can be achieved with blocking Abs, but also by inhibiting the enzyme glutaminyl cyclase (QC) with small molecules. Glutaminyl cyclase inhibition reduces N‐terminal pyro‐glutamate formation of CD47 at the SIRPα binding site. Here, we investigated the impact of QC inhibition on myeloid effector cell‐mediated tumor cell killing by epidermal growth factor receptor (EGFR) Abs and the influence of Ab isotypes. SEN177 is a QC inhibitor and did not interfere with EGFR Ab‐mediated direct growth inhibition, complement‐dependent cytotoxicity, or Ab‐dependent cell‐mediated cytotoxicity (ADCC) by mononuclear cells. However, binding of a human soluble SIRPα‐Fc fusion protein to SEN177 treated cancer cells was significantly reduced in a dose‐dependent manner, suggesting that pyro‐glutamate formation of CD47 was affected. Glutaminyl cyclase inhibition in tumor cells translated into enhanced Ab‐dependent cellular phagocytosis by macrophages and enhanced ADCC by polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophilic granulocyte‐mediated ADCC was significantly more effective with EGFR Abs of human IgG2 or IgA2 isotypes than with IgG1 Abs, proposing that the selection of Ab isotypes could critically affect the efficacy of Ab therapy in the presence of QC inhibition. Importantly, QC inhibition also enhanced the therapeutic efficacy of EGFR Abs in vivo. Together, these results suggest a novel approach to specifically enhance myeloid effector cell‐mediated efficacy of EGFR Abs by orally applicable small molecule QC inhibitors.

Inhibition of Arginase 1 Liberates Potent T Cell Immunostimulatory Activity of Human Neutrophil Granulocytes.

Myeloid cell arginase-mediated arginine depletion with consecutive inhibition of T cell functions is a key component of tumor immune escape. Both, granulocytic myeloid-derived suppressor cells (G-MDSC) and conventional mature human polymorphonuclear neutrophil granulocytes (PMN) express high levels of arginase 1 and can act as suppressor cells of adaptive anti-cancer immunity. Here we demonstrate that pharmacological inhibition of PMN-derived arginase 1 not only prevents the suppression of T cell functions but rather leads to a strong hyperactivation of T cells. Human PMN were incubated in cell culture medium in the absence or presence of an arginase inhibitor. T cells from healthy donors were then activated either polyclonally or in an antigen-specific manner in the supernatants of the PMN cultures at different PMN-T cell ratios. T cell proliferation was completely suppressed in these supernatants in the absence of an arginase inhibitor. Arginase inhibition led to a strong hyperinduction of T cell proliferation, which exceeded control activation conditions up to 25-fold. The hyperinduction was correlated with higher PMN-T cell ratios and was only apparent when PMN arginase activity was blocked sufficiently. The T cell stimulatory factor was liberated very early by PMN and was present in the < 3 kDa fraction of the PMN supernatants. Increased T cell production of specific proinflammatory cytokines by PMN supernatant in the presence of arginase inhibitor was apparent. Upon arginase inhibition, downregulation of important T cell membrane activation and costimulation proteins was completely prevented or de novo induction accelerated. Antigen-specific T cell cytotoxicity against tumor cells was enhanced by PMN supernatant itself and could be further increased by PMN arginase blockade. Finally, we analyzed anergic T cells from multiple myeloma patients and noticed a complete reversal of anergy and the induction of strong proliferation upon T cell activation in PMN supernatants by arginase inhibition. In summary, we discovered a potent PMN-mediated hyperactivation of human T cells, which is apparent only when PMN arginase-mediated arginine depletion is concurrently inhibited. Our findings are clearly relevant for the analysis and prevention of human tumor immune escape in conjunction with the application of arginase inhibitors already being developed clinically.

α9α10 nicotinic acetylcholine receptors regulate murine bone marrow granulocyte functions

Polymorphonuclear neutrophilic granulocytes (PMNs) are extremely important in defense of the organism against infections and in inflammatory processes including neuroinflammation and pain sensation. Different subtypes of nicotinic acetylcholine receptors (nAChRs) are involved in modulation of PMN activities. Earlier we determined expression of α2-7, α9, β3, β4 subunits and regulatory role of α7 and α3β2 nAChR subtypes in functions of inflammatory PMNs. Other authors detected mRNA of α9 subunit in bone marrow neutrophils (BM-PMNs). Murine BM-PMNs coming out from the bone marrow, where they develop, to blood were characterized as mature. There was no data for α10 and for the presence of functionally active α9α10 nAChRs in BM-PMNs. Here we detected for the first time mRNA expression of the α10 nAChR subunit in BM-PMNs and confirmed the expression of mRNA for α9 nAChR. With the help of α-conotoxins RgIA and Vc1.1, highly selective antagonists of α9α10 nAChRs, we have revealed participation of α9 and/or α9α10 nAChRs in regulation of cytosolic Ca2+ concentration, cell adhesion, and in generation of reactive oxygen species (ROS). Nicotine, choline, RgIA, and Vc1.1 induced Ca2+ transients in BM-PMNs, enhanced cell adhesiveness and decreased production of ROS indicating involvement of α9, possibly co-assembled with α10, nAChRs in the BM-PMN activity for recruitment and cytotoxicity.

Neutrophil Effector Responses to Cystic Fibrosis Clinical Isolates of Staphylococcus aureus and Pseudomonas aeruginosa

Abstract Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of polymorphonuclear neutrophil granulocytes (PMNs). In CF, PMNs are unable to clear lung pathogens resulting in lung damage. Neutrophil extracellular traps (NETs) and superoxide production represent defense mechanisms of PMNs to kill extracellular pathogens. NETs and superoxide in the CF lung, however, seem unable to clear Pseudomonas aeruginosa and Staphylococcus aureus, the two main lung pathogens in CF. Our goal of this study was to determine antimicrobial functions of PMNs to S. aureus and P. aeruginosa CF isolates and to observe how the CF airway environment influences them. Human PMNs were isolated from healthy donors. Isolates of S. aureus were obtained from adult CF patients: four strains were methicillin-resistant (MRSA), four were methicillin-sensitive S. aureus (MSSA), and ten CF isolates of P. aeruginosa. PMN effector functions were measured: killing was assessed by bacterial growth assay, NET formation was measured by Sytox fluorescence and superoxide was monitored by Diogenes chemiluminescence. Results were subjected to paired t-test, significant if p&amp;lt;0.05. PMNs from healthy volunteers were able to kill all CF isolates. The extent of killing varied among strains, however, P. aeruginosa was killed more efficiently by blood PMNs than S. aureus clinical isolates. The enhanced killing of P. aeruginosa is correlated with enhanced NET release and superoxide production compared to S. aureus. Killing of all isolates was inhibited by CF sputum pretreatment. Overall, the results show that CF bacterial clinical isolates stimulate PMN effector responses that are impaired by the CF airway environment.

15-Epi-LXA4 and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA4 or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA4 and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.

Immunobiological status of the body of cows during mastitis

The work reveals the immunobiological aspects of lactation of cows and changes in immunobiological reactivity in the development of mastitis.The authors present modern scientific data on the local immune protection of the mammary gland of cows. Main stages of ontogenetic development of cellular immunity of the mammary gland of cows were traced during clinical and experimental studies. The number of somatic cells in the secret of the mammary gland of the primates was dependent on the period of the functioning of the mammary gland. In the cytology of colostrum mostly (56.00 ± 1.90%) neutrophil granulocytes were predominant, in the middle period of lactation (3–5th month) the proportion of epithelial cells increased (from 29.51 ± 2.17 to 49.59 ± 1.94%), during the launch period, the population of polymorphonuclear neutrophil granulocytes was changing as well, which virtually recovered to the original level and increased during the dry period. However, at the end of lactation, during the onset and dry, with the development of involutionary processes in the mammary gland, a sharp decrease in cytochemical reactivity of intracellular lysozyme of phagocytic cells was observed. To conduct clinical and experimental studies, three groups of animals were formed. As a result, it was found out that subclinical mastitis of cows is accompanied by a change in the immunobiological reactivity. Purulent-catarrhal mastitis in cows was manifested by significant changes in the parameters of nonspecific immunological reactivity. In the peripheral blood of cows with subclinical mastitis, the number of reactive microphages increased sharply (P &lt; 0.001). In parallel with this, the number of activated phagocytes with myeloperoxidase granules also increased in the peripheral blood (P &lt; 0.01). Activation of intra-leukocyte lysozyme phagocytic cells was less intensive. Subclinical udder pathology was accompanied by an increase in the number of degranulated cells (P &lt; 0.001), which is one of the specific properties of cytomorphological changes in programmed death (apoptosis). Subclinical inflammation of the mammary glands mastitis of cows was accompanied by a certain decrease in the number of T-lymphocytes (P &lt; 0.001). Clinical and experimental studies have shown that subclinical and purulent-catarrhal mastitis of cows undergo significant changes in systemic immunity. In the pathophysiological model of subclinical and purulent-catarrhal mastitis, the functional state of the T-link of specific immunity was disturbed, the bactericidal activity of blood serum and phagocytosis were suppressed, which occurred against the background of changes in the cytochemical reactivity of phagocytic cells circulating immune complexes and molecules with an average molecular weigh.

Role of polymorphonuclear neutrophils in the reperfused ischemic brain: insights from cell-type-specific immunodepletion and fluorescence microscopy studies.

Polymorphonuclear neutrophil granulocytes (PMNs) are part of the early post-ischemic immune response that orchestrates the removal of infarcted brain tissue. PMNs contribute to secondary brain injury in experimental stroke models. In human patients, high PMN-to-lymphocyte ratios in peripheral blood are predictive of poor stroke outcome. Following earlier studies indicating that the cerebral microvasculature forms an efficient barrier that impedes PMN brain entry even under conditions of ischemia, more recent studies combining intravital two-photon microscopy and ex vivo immunohistochemistry unequivocally demonstrated the accumulation of PMNs in the ischemic brain parenchyma. In the meantime, transgenic mouse lines, such as mice expressing Cre-recombinase and the red fluorescent reporter protein tdTomato under the highly granulocyte-specific locus for the gene Ly6G (so-called Catchup mice), have become available that allow study of dynamic interactions of PMNs with brain parenchymal cells. These mice will further help us understand how PMNs promote brain injury and disturb brain remodeling and plasticity.

Онтогенетичні особливості формування локального імунного захисту молочної залози корів (огляд літератури та оригінальні дослідження)

The authors present modern scientific data on the local immune protection of the mammary gland of cows. Main stages of ontogenetic development of cellular immunity of the mammary gland of cows were traced during clinical and experimental studies. The number of somatic cells in the secret of the mammary gland of the primates was dependent on the period of the functioning of the mammary gland. In the cytology of colostrum mostly (56.00 ± 1.90%) neutrophil granulocytes were predominant, in the middle period of lactation (3–5th month) the proportion of epithelial cells increased (from 29.51 ± 2.17 to 49.59 ± 1.94%), during the launch period, the population of polymorphonuclear neutrophil granulocytes was changing as well, which virtually recovered to the original level and increased during the dry period. The cytochemical reactivity of intracellular lysozyme of phagocytic cells in the secretion of the breast of the primates was from the beginning of lactogenesis and in the middle period of lactation (3-5th month) almost constant. However, at the end of lactation, during the onset and dry, with the development of involutionary processes in the mammary gland, a sharp decrease in cytochemical reactivity of intracellular lysozyme of phagocytic cells was observed. A similar trend was observed in the phagosomal activity of lysosomal cationic proteins. The greatest reactivity of phagocytes was shown in the beginning of lactogenesis in reaction to lysosomal cationic proteins, which acquired its maximum manifestation in the middle period of lactation and gradually decreased at the end of the lactation period. In the period of launch and dry in the secretion of pricking cessation, there was a cytochemical activation of oxigen-dependent factors of protection of phagocytic cells. Consequently, the formation of cellular immune defense takes place in the process of ontogenetic development of the mammary gland of cows. In the firstborn, simultaneously with the formation of secretory function of the mammary gland, there is a gradual formation of natural factors protecting the body. Oxygen-independent and Oxygen-dependent factors of phagocytic protection of the breast of the firstborn are not sufficiently formed, their activation is started from the colostrum period and undergoes a physiological fluctuation throughout the lactation period.

Interactions between Neutrophils and Pseudomonas aeruginosa in Cystic Fibrosis.

Cystic fibrosis (CF) affects 70,000 patients worldwide. Morbidity and mortality in CF is largely caused by lung complications due to the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Cystic fibrosis airway inflammation is mediated by robust infiltration of polymorphonuclear neutrophil granulocytes (PMNs, neutrophils). Neutrophils are not capable of clearing lung infections and contribute to tissue damage by releasing their dangerous cargo. Pseudomonas aeruginosa is an opportunistic pathogen causing infections in immunocompromised individuals. P. aeruginosa is a main respiratory pathogen in CF infecting most patients. Although PMNs are key to attack and clear P. aeruginosa in immunocompetent individuals, PMNs fail to do so in CF. Understanding why neutrophils cannot clear P. aeruginosa in CF is essential to design novel therapies. This review provides an overview of the antimicrobial mechanisms by which PMNs attack and eliminate P. aeruginosa. It also summarizes current advances in our understanding of why PMNs are incapable of clearing P. aeruginosa and how this bacterium adapts to and resists PMN-mediated killing in the airways of CF patients chronically infected with P. aeruginosa.

Neutrophil extracellular traps in health and disease

Polymorphonuclear neutrophil granulocytes are the first responders of the immune system to threats by invading microorganisms. In the traditional view, they combat the intruders by phagocytosis and externalisation of granules containing lytic and microbicidal factors. A dozen years ago, this concept was expanded by the observation that neutrophils may react to bacteria by extruding their nuclear chromosomal DNA with attached nuclear and cytoplasmic constituents to form extracellular reticular structures. Since they trapped and immobilised the microbes, they were designated neutrophil extracellular traps (NETs), and their ensuing cell death NETosis. Subsequently, the NETs were shown to act against different types of pathogens, including viruses, and an intricate interplay between the NETs and countermeasures of the pathogens became apparent. The NETs were also found to induce inflammatory responses in the host that contributed to the pathophysiology of autoinflammatory and even autoimmune diseases. Of special interest is the direct link that NETs provide to infections that may initiate and maintain inflammation without the participation of adaptive immunity. In contrast, neutrophils seem capable of activating B cells to produce antibodies relevant to autoimmunity independently of T cell help. Further results imply NETs in the occurrence of thrombosis of the veins and recently also in the generation of arterial plaque. Data from the studies on the defence against pathogens and the pathophysiology of inflammation and thrombosis have started to drive applications to modulate NET formation and its effects and may provide opportunities to optimise current diagnostic and therapeutic concepts.

The clinical presentation and therapy of diseases related to anti-neutrophil cytoplasmic antibodies (ANCA).

Anti-neutrophil cytoplasmic antibodies (ANCA) are a family of autoantibodies that react with proteins predominantly expressed in cytoplasmic granules of polymorphonuclear neutrophil granulocytes (PMNs). ANCA was initially detected using indirect immunofluorescence, allowing for different patterns such as p-ANCA (perinuclear) and c-ANCA (cytoplasmic) to be distinguished. Today it is common to detect the antibodies by immunochemical assays such as ELISA using purified proteins as antigens. The strongest association with ANCA is found in the pauci-immune small vessel vasculitides granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). There is compelling evidence that ANCA contributes to the pathogenesis in these conditions. ANCA also occurs in 30%-40% of patients with eosinophilic granulomatosis with polyangiitis (EGPA) and anti-GBM disease, but is uncommon in other forms of vasculitis. ANCA with different specificities have been described with varying frequencies in diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, inflammatory bowel disease, endocarditis, chronic infections and hematopoietic malignancies. ANCA can also develop as an adverse event during pharmacological treatment. These entities are treated quite differently, with therapies ranging from immunosuppressive agents over antibiotics to simply removing the causative drug. A positive ANCA test thus requires a careful diagnostic work-up.

Effect of salivary agglutination on oral streptococcal clearance by human polymorphonuclear neutrophil granulocytes

Salivary agglutination is an important host defense mechanism to aggregate oral commensal bacteria as well as invading pathogens. Saliva flow and subsequent swallowing more easily clear aggregated bacteria compared with single cells. Phagocytic clearance of bacteria through polymorphonuclear neutrophil granulocytes also seems to increase to a certain extent with the size of bacterial aggregates. To determine a connection between salivary agglutination and the host innate immune response by phagocytosis, an invitro agglutination assay was developed reproducing the average size of salivary bacterial aggregates. Using the oral commensal Streptococcus gordonii as a model organism, the effect of salivary agglutination on phagocytic clearance through polymorphonuclear neutrophil granulocytes was investigated. Here we describe how salivary aggregates of S.gordonii are readily cleared through phagocytosis, whereas single bacterial cells showed a significant delay in being phagocytosed and killed. Furthermore, before phagocytosis the polymorphonuclear neutrophil granulocytes were able to induce a specific de-aggregation, which was dependent on serine protease activity. The data presented suggest that salivary agglutination of bacterial cells leads to an ideal size for recognition by polymorphonuclear neutrophil granulocytes. As a first line of defense, these phagocytic cells are able to recognize the aggregates and de-aggregate them via serine proteases to a more manageable size for efficient phagocytosis and subsequent killing in the phagolysosome. This observed mechanism not only prevents the rapid spreading of oral bacterial cells while entering the bloodstream but would also avoid degranulation of involved polymorphonuclear neutrophil granulocytes, so preventing collateral damage to nearby tissue.

Polymorphonuclear neutrophils in periodontitis and their possible modulation as a therapeutic approach.

The main focus of this review is polymorphonuclear neutrophilic granulocytes. Polymorphonuclear neutrophils play a pivotal role in normal host resistance to subgingival dental-plaque biofilm. Both hyper- and hypo-responsiveness of the immune system toward the microbial challenge in periodontitis have been described. We review polymorphonuclear neutrophil physiology with emphasis on the role of neutrophil functions and dysfunctions in periodontitis. Text boxes are given at the end of each subsection, which present the current knowledge on neutrophil-modulating agents as a potential therapeutic approach in periodontitis.

Soft Tissue Response to Titanium Abutments with Different Surface Treatment: Preliminary Histologic Report of a Randomized Controlled Trial.

The aim of this preliminary prospective RCT was to histologically evaluate peri-implant soft tissues around titanium abutments treated using different cleaning methods. Sixteen patients were randomized into three groups: laboratory customized abutments underwent Plasma of Argon treatment (Plasma Group), laboratory customized abutments underwent cleaning by steam (Steam Group), and abutments were used as they came from industry (Control Group). Seven days after the second surgery, soft tissues around abutments were harvested. Samples were histologically analyzed. Soft tissues surrounding Plasma Group abutments predominantly showed diffuse chronic infiltrate, almost no acute infiltrate, with presence of few polymorphonuclear neutrophil granulocytes, and a diffuse presence of collagenization bands. Similarly, in Steam Group, the histological analysis showed a high variability of inflammatory expression factors. Tissues harvested from Control Group showed presence of few neutrophil granulocytes, moderate presence of lymphocytes, and diffuse collagenization bands in some sections, while they showed absence of acute infiltrate in 40% of sections. However, no statistical difference was found among the tested groups for each parameter (p > 0.05). Within the limit of the present study, results showed no statistically significant difference concerning inflammation and healing tendency between test and control groups.