Polymorph Nuclear Neutrophils Research Articles

The serum-ascites vitamin D gradient: a diagnostic marker for spontaneous bacterial peritonitis in Egyptian cirrhotic patients

Background The most prevalent infectious consequence of liver cirrhosis is spontaneous bacterial peritonitis (SBP). Ascitic fluid polymorph nuclear neutrophils (PMN) counts of more than 250 cells/µL are used as the gold standard for diagnosing SBP. Patients with decompensated cirrhosis also had significantly decreased serum 25-OH vitamin D levels compared with controls. Aim Our aim is to investigate the role of (serum ascites vitamin D gradient) as a diagnostic factor for diagnosis of SBP. Patients and method This cross-sectional study included 80 cirrhotic ascitic patients from Egypt who were treated at the hospitals affiliated with the Tropical Medicine and Infectious Diseases Department at Tanta University. Group 1: 40 ascitic patients with SBP. Group 2: 40 ascitic patients without SBP History taking, clinical examination, laboratory analysis, and imaging studies were applied to every patient. Results Child-Pugh score was significant in both groups as child C was significantly higher in group 1 than group 2. Total leucocyte count and differential neutrophil were significantly higher in group 1 than group 2 while child B was higher in group 2 than group 1. Differential lymphocyte was significantly lower in group 1 than group 2. SADG was significantly lower in group 1 than group 2. SADG ratio can significantly predict SBP (P = 0.036 and AUC = 0.632) at cut-off less than or equal to −25.8 with 37.50% sensitivity, 95.00% specificity, 88.2% positive predictive value (PPV), and 60.3% negative predictive value (NPV). Conclusion Patients with cirrhotic ascites had decreased SADG and higher SBP. In these cases, it might be used as a diagnostic and prognostic marker for SBP. Patients with decompensated cirrhosis, independent of SBP, have an increased prevalence of vitamin D insufficiency.

In vivo dynamics of pro-inflammatory factors, mucins, and polymorph nuclear neutrophils in the bovine oviduct during the follicular and luteal phase

Dynamic functional changes in the oviductal microenvironment are the prerequisite for the establishment of pregnancy. The objective of this study was to gain the first insights into oestrous cycle-dependent dynamics of polymorph nuclear neutrophils (PMN) and the mRNA abundance of selected genes and their correlations in the oviduct of living cows. Mini-cytobrush samples were taken from the oviducts of healthy heifers (n = 6) and cows (n = 7) during the follicular (FOL) and luteal phase (LUT) by transvaginal endoscopy. Total RNA was isolated from the samples and subjected to reverse transcription-quantitative PCR for selected pro-inflammatory factors, glycoproteins, and a metabolic marker. The percentage of PMN was determined by cytological examination. The mean PMN percentage was 2.8-fold greater during LUT than FOL. During LUT, significantly greater mRNA abundance of the pro-inflammatory factors IL1B, CXCL1, CXCL3, and CXCL8 was observed. The OVGP1 mRNA abundance was twice as high during FOL than in LUT. Pearson correlation, principal component analysis and heatmap analyses indicated characteristic functional patterns with strong correlations among investigated factors. Using this novel approach, we illustrate complex physiological dynamics and interactions of the mRNA expression of pro-inflammatory factors, mucins, OVGP1, and PMN in the oviduct during the oestrous cycle.

The effect of smoking in the polymorph nuclear neutrophils’ functions in patients with gingivitis

Smoking is a habit that is widespread in today’s world as its primary constituent called Nicotine makes it very addictive for the users. Smoking is an important cause of diseases related to gums. If not quitted, it may lead to progressive adverse conditions in the oral cavity like gingivitis, periodontitis and so on. This is because the functions of neutrophils are significantly affected by smoking. As a result of the suppression of the activity of the neutrophils, the overall oral health of the individuals also become compromised. This article aims to describe the pattern and clearly delineate the difference in the condition of gingiva between smokers and nonsmokers by explaining the changes brought about by smoking on the polymorphonuclear neutrophils. Three groups of people were selected for this study. The groups were matched by age and gingival condition. Two groups consisted of people with the habit of smoking, and they were further categorized into mild smokers and heavy smokers depending on the number of cigarettes smoked per day. The third group consisted of people with no habit of smoking. This group was the control group in the study. Blood samples were taken and Polymorphonuclear neutrophils were isolated. The functions of PMNs like chemotaxis and phagocytosis were studies under different methods. The mean value for the all the three groups under clinical parameters like plaque index, gingival index, and the functions of neutrophils like chemotaxis and phagocytosis are obtained and after elucidating the results, it is found that smoking impairs the overall oral hygiene and the functions of neutrophils against gingival infections.It is proven that there is debasing alteration in the functions of neutrophils which could have led to the localized destruction. Hence it suggests that smoking impairs the functions of neutrophils against gingival infections. And there may be a higher possibility of this condition to progress into the next stage called periodontitis. Hence it is advisable to quit the habit as and when it is possible, and it is the role of dentists to identify such conditions and educate the patients on the adverse effects of smoking.

Evaluation of Efficacy and Potential Complications of the Plant-Based Nanoparticles Hemostat Powder on Controlling Hemorrhage in Rat Models of Liver Injury: An Experimental Study

BackgroundLiver surgery is associated with an increased risk of bleeding, which required time management. Several hemostatic agents have been developed. However, plant-based materials have shown some superiority in hemostasis time and lower complications. The aim of our study was to prepare plant-based topical hemostatic Nanoparticles from the rosaceous family as a novel hemostatic agent.Material and Methods Twenty male Wistar rats were randomly divided into two study groups. All the rats were sedated and underwent incision in the anterior lobe of the liver. In the control group, the incision site was firstly compressed with a manual compression with dry gauze for 2 minutes and then repeated every 30 seconds until hemostasis was achieved. In the intervention group, homeostatic material was applied on the site of the incision, and clotting time was recorded. Paired Samples Test was used to compare the mean hemo­stasis times in Nanoparticles and control groups. P <0.05 considered significant.ResultsHemostasis was successfully achieved in both groups. Hemostasis time in Nanoparticles and control groups were 6.7±1.33 and 183±26.26 seconds, respectively and their differences were significant (P < 0.001). The histopathology assessment showed mild increases in vascularity, fibrosis, and polymorph nuclear neutrophils (PMN) infiltration was seen in one case. Mild to moderate lymphocyte infiltration was detected in 2 cases. ConclusionThe produced plant-based Nanoparticles could significantly reduce the hemostasis time compared to the control group.

Value of Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) as a Diagnostic Marker for Neonatal Sepsis

Introduction: Neonatal Sepsis (NS) is a serious clinical condition caused by a dysregulated immune response to an infection. Neonatal period is the most vulnerable time in the child’s life. Globally, approximately 7000 newborns die everyday. Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) is an important receptor for the inflammatory response regulated by neutrophils. Aim: To explore the role of TREM-1 as a potential early diagnostic marker of NS compared to the conventional blood culture technique. The prognostic utility and relation of TREM-1 expression level to the clinical disease severity in septic neonates were also evaluated. Materials and Methods: The study enrolled 75 neonates with NS admitted to the Neonatal Intensive Care Unit (NICU). The studied neonates were categorised into three groups; group I: 25 neonates with an Early Onset Sepsis (EOS), group II: 25 neonates with a Late Onset Sepsis (LOS), group III: 25 neonates with septic shock. In addition, 25 age and sex-matched healthy neonates with no evidence of sepsis or any other medical illness were studied as a control group. Blood samples for conventional blood cultures and estimation of TREM-1 gene expression level in Polymorph Nuclear Neutrophils (PMNs) using quantitative Real-Time Polymerase Chain Reaction (RT-PCR) assay were synchronously collected. Data regarding clinical and laboratory findings, and risk factors of NS were also analysed. Statistical Package for the Social Sciences (SPSS) software version 20 was used to analyse the data obtained from the study. Results: The total culture-proven cases represented 24% (18/75) of all the studied neonates with NS. Klebsiella spp. was the most frequently isolated Gram-negative bacteria (4/18; 22.2%) followed by Pseudomonas spp. (3/18; 16.7%) and Acinetobacter spp. (1/18; 5.5%). Coagulase- Negative Staphylococci (CoNs), S. aureus and Candida spp. accounted for 22.2% (4/18), 16.7% (3/18) and 16.7% (3/18) of the isolated organisms respectively. No statistically significant difference was detected between the three studied groups as regards blood culture results. Significant statistical differences were detected between groups I, II and III in relation to the control group (p=0.048, p=0.049 and p&lt;0.001) regarding TREM-1 mRNA expression level. Low Birth Weight (LBW) and prematurity were the most significant risk factors for NS. At a cut-off point of ≥0.631 TREM-1 mRNA could be considered as a potential marker for diagnosis of NS with sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of 65.33%, 96.0%, 98.0% and 48.0%, respectively. The prognostic utility of TREM-1 gene expression proved a sensitivity of 87.8% at a cut-off point &gt;0.369. Conclusion: TREM-1 gene expression has a potential value in prognostic assessment of NS and could be considered as an early diagnostic marker.

Fibrinogen and Fibrin Differentially Regulate the Local Hydrodynamic Environment in Neutrophil–Tumor Cell–Endothelial Cell Adhesion System

As cancer is one of the major fatal diseases for human beings worldwide, the metastasis of tumor cells (TCs) from a blood vessel to an adjacent organ has become a focus of research. A tumor metastasis theory named the “two-step theory” pointed out that polymorphnuclear neutrophils (PMNs) could facilitate TC adhesion on an endothelial monolayer under flow, which was regulated by shear flow and promoted by fibrinogen and fibrin. In order to further understand the role of hydrodynamics played in the “two-step theory”, we improved our side-view micro-particle imaging velocimetry (PIV) system and successfully measured the flow velocity profiles around adherent PMNs and TCs on an endothelial monolayer in the presence of soluble fibrinogen or fibrin under shear flow. Combined with a computational fluid dynamics simulation, we found that: (1) soluble fibrinogen and fibrin influenced the variations of relative shear rates above an adhered PMN and an adherent TC at different PMN-to-TC position states; (2) compared with soluble fibrinogen, soluble fibrin made the curves of relative shear rates above an adherent cell flatter. Soluble fibrin might increase the collision frequency and affect the contact time and contact area between PMNs, TCs, and endothelium cells, resulting in the enhancement of TC adhesion and retention on an endothelial monolayer.

Role of adenosine A2b receptors in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide

To explore the role of the low-affinity A2b adenosine receptors (Adora2b) in pulmonary microvascular endothelial inflammation induced by lipopolysaccharide and its mechanism. Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and cultured in vitro. After serum deprivation for 24 hours, cells were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then challenged with LPS (100 μg/L). Cells without treatment were served as the control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. After incubation with specific drugs for 24 hours, the apoptosis of PMVECs was analyzed by flow cytometry using Annexin V/propidium iodide (PI) technique. The levels of early inflammatory factors in cultured medium were measured using enzyme linked immunosorbent assay (ELISA). The mRNA expressions of chemotactic factors and adhesion molecules were determined by real-time quantitative-polymerase chain reaction (RT-qPCR). Polymorph nuclear neutrophils (PMNs) from venous blood of healthy rats were isolated, and PMN migration through PMVECs monolayer under stimulation of drugs was observed in transwell inserts. The monolayer permeability of PMVECs after adhesion of PMNs was determined by fluorescein isothiocyanate (FITC)-albumin assay. Oxidative stress was detected by DCFH-DA assay. Compared with the control group, more cells entered into the apoptosis stage after LPS challenge. Meanwhile, the levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in cultured medium were significantly increased, as well as the mRNA expressions of chemotactic factors [C-X-C motif chemokine ligand 1 (CXCL-1), CXCL-3 and monocyte chemoattractant protein-1 (MCP-1)] and adhesion molecules [E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. More PMNs migrated through PMVECs following adhesion and the monolayer permeability of PMVECs was rapidly enhanced. The oxidative stress was upregulated. Compared with LPS group, BAY60-6583 pretreatment could dose-dependently decrease the rate of apoptosis, attenuate trans-endothelial migration of PMNs and decrease the endothelial cell barrier leakage. There were significant differences even after incubation of 0.1 μmol/L BAY60-6583 [apoptosis rate: (21.12±2.12)% vs. (27.66±3.57)%, number of migrated PMNs/HP: 260.60±18.24 vs. 290.20±16.48, permeability coefficient (Pd, ×10-6 cm/s): 28.28±2.04 vs. 32.55±2.13, all P < 0.05]. Meanwhile, BAY60-6583 pretreatment also downregulated the levels of early proinflammatory factors in a dose-dependent manner as well as the mRNA expressions of chemotactic factors and adhesion molecules. The statistic difference was significant while treated with 1 μmol/L BAY60-6583 [IL-1β (ng/L): 475.75±63.15 vs. 755.25±67.42, TNF-α (ng/L): 560.25±69.96 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 3.57±0.28 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 4.56±0.48 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 2.21±0.31 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 4.64±0.09 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 4.14±0.30 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 2.23±0.19 vs. 2.92±0.33, all P < 0.05]. Furthermore, pretreatment of 10 μmol/L BAY60-6583 could decrease the oxidative stress [reactive oxygen species (RFU): 629.05±33.10 vs. 781.45±64.59, P < 0.05]. Contrast, PSB1115 pretreatment aggravated apoptosis of PMVECs after LPS incubation [(34.36±4.57)% vs. (27.66±3.57)%], upregulated expressions of proinflammatory and chemotactic factors as well as adhesion molecules [IL-1β (ng/L): 889.00±63.11 vs. 755.25±67.42, TNF-α (ng/L): 939.00±43.44 vs. 818.75±60.92, CXCL-1 mRNA (2-ΔΔCt): 6.66±0.65 vs. 5.27±0.69, CXCL-3 mRNA (2-ΔΔCt): 10.42±0.51 vs. 7.32±0.54, MCP-1 mRNA (2-ΔΔCt): 4.85±0.34 vs. 3.35±0.21, E-selectin mRNA (2-ΔΔCt): 8.42±0.47 vs. 7.28±0.73, ICAM-1 mRNA (2-ΔΔCt): 7.46±0.72 vs. 5.89±0.25, VCAM-1 mRNA (2-ΔΔCt): 4.35±0.26 vs. 2.92±0.33], aggravated trans-endothelial migration of PMNs (cells/HP: 348.40±22.68 vs. 290.20±16.48), enhanced the leakage of PMVECs monolayer [Pd (×10-6 cm/s): 39.65±2.69 vs. 32.55±2.13] and increased oxidative stress in PMVECs [reactive oxygen species (RFU): 847.04±29.26 vs. 781.45±64.59], with statistically significant difference (all P < 0.05). Activation of endothelial Adora2b attenuates LPS-induced pulmonary microvascular inflammation by decreasing the release of early inflammatory factors, downregulating expressions of chemotactic factors and adhesion molecules, attenuating trans-endothelial migration of PMNs and oxidative stress in PMVECs, which suggest endothelial Adora2b is apotential anti-inflammatory target in the treatment of LPS-induced acute lung injury.

Abstract 1100: Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma

Abstract The GBM microenvironment is composed of numerous non-neoplastic cells, among which tumor-associated myeloid cells (TAMs) are the most abundant. TAMs are constituted of several different cell types, including brain microglia and bone-marrow-derived infiltrating cells, which can be further divided into inflammatory monocytes, monocyte-derived macrophages, and polymorphnuclear neutrophils. These myeloid cells are believed to promote tumor growth and immune evasion by expressing immune checkpoint proteins. Remarkable progress has been made in treating lung cancer and melanoma by using immune-checkpoint inhibitors; however, mixed results were obtained for its application in GBM. To examine how molecular-subtypes of GBM influence the composition and immunosuppression of tumor-associated myeloid cells, we used the RCAS_tv-a system, a somatic cell-specific gene transfer system, to generate de novo murine GBM (mGBM). By manipulating different known human oncogenic-driver mutations such as overexpressing PDGFB or EGFRVIII, or silencing NF1, we generated Proneural (PN), Classical (CL) and Mesenchymal (MES) GBMs respectively, which phenotypically resemble their human counterparts. We found that the majority (80%) of tumor-infiltrating myeloid cells in PN GBM are bone-marrow derived macrophages, whereas this number is significantly less in CL and MES mGBM. Interestingly, unique to MES, there is a significant presence (16%) of neutrophils in these tumors than in other subtypes (4%). Further characterization for subtype-specific expression of PD-L1, a druggable immune checkpoint molecule, by flow cytometry shows that MES has the highest expression of PD-L1), whereas the expression is lower in PN and CL subtypes. Treating GBM-bearing mice with anti-PDL1 antibodies did not prolong their survival. Concurrent treatment of irradiation and anti-PDL1 antibody did not confer significant survival advantage either compared to controls. These results reflect the unsuccessful trails of recent human clinical studies; however, it also emphasizes the importance of understanding the mechanism of resistance in GBM immunotherapy before effective immune treatment can be established. Our immunocompetent mouse models provide indispensable tools for these studies. Note: This abstract was not presented at the meeting. Citation Format: Zhihong Chen, Cameron J. Hurting, Ben Gabanic, Jyothi Murukuti, Dolores Hambardzumyan. Genetic driver-mutations define composition and properties of tumor-associated myeloid cells in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1100.

ФАГОЦИТАРНА АКТИВНІСТЬ НЕЙТРОФІЛІВ У ХВОРИХ НА ГОСТРИЙ ГЕПАТИТ В

Проникаючи в організм, інфекційні агенти стикаються з вродженими імунними клітинами, серед яких фагоцити знаходяться на першій лінії захисту.Мета роботи – вивчення функціонального стану поліморфноядерних нейтрофілів у крові хворих на гострий гепатит В (ГГВ) різного ступеня тяжкості у динаміці загальноприйнятого лікування та після призначення симбіотика. Пацієнти і методи. Під спостереженням перебувало 108 хворих на ГГВ, від 18 до 69 років (середній вік − 34,0±1,9 роки), з них 56 чоловіків і 52 жінки. Конт­рольну групу склали 17 здорових осіб. Для оцінки фагоцитарної активності нейтрофілів (ФАН) визначали фагоцитарний індекс нейтрофілів (ФІН) і фагоцитарне число нейтрофілів (ФЧН) з періодами інкубації 30, 60 і 180 хв.Висновки. Виявлено значні компенсаторні можливості нейтрофілів, які здатні в умовах інтенсивного HBV навантаження підтримувати загальний баланс у фагоцитарній системі. Включення симбіотика до спільної схеми терапії підвищує як інтенсивність фагоцитозу, так і регулює поглинаючі можливості нейтрофілів у хворих на HBV-інфекцію різного ступеня тяжкості.

Increase of CD25 expression on bovine neutrophils correlates with disease severity in post-partum and early lactating dairy cows

Polymorph-nuclear neutrophils (PMN) in cattle exhibit unique features when compared to human or murine PMN and are of particular interest concerning the risk of post-partum mammary gland or extra-mammary infections related to the periparturient suppression of neutrophil functions. Former studies could show that effects of IL-2 on innate immune cells such as PMN were mediated by the interleukin-2 receptor (IL-2R) β and γ chains. In the current study we could detect IL-2Rα (CD25) expression on bovine PMN using flow-cytometric analysis. CD25 was detected on granulocytes from post-partum and early lactating cows with different inflammatory conditions. The expression of CD25 on PMN in blood and raw milk increased with disease severity. Our results suggest CD25 expression on PMN as a potential biomarker for acute infections in cattle. Furthermore, our data provide a basis to better understanding of the periparturient functional suppressions of PMN that might reveal new molecular targets for therapy or prevention of disease.

Inflammatory responses following intramuscular and subcutaneous immunization with aluminum-adjuvanted or non-adjuvanted vaccines

Aluminum-adjuvanted vaccines are administered through an intramuscular injection (IM) in the US and EU, however, a subcutaneous injection (SC) has been recommended in Japan because of serious muscle contracture previously reported following multiple IMs of antibiotics. Newly introduced adjuvanted vaccines, such as the human papillomavirus (HPV) vaccines, have been recommended through IM. In the present study, currently available vaccines were evaluated through IM in mice. Aluminum-adjuvanted vaccines induced inflammatory nodules at the injection site, which expanded into the intra-muscular space without any muscle degeneration or necrosis, whereas non-adjuvanted vaccines did not. These nodules consisted of polymorph nuclear neutrophils with some eosinophils within the initial 48h, then monocytes/macrophages 1 month later. Inflammatory nodules were observed 6 months after IM, had decreased in size, and were absorbed 12 months after IM, which was earlier than that after SC. Cytokine production was examined in the injected muscular tissues and AS04 adjuvanted HPV induced higher IL-1β, IL-6, KC, MIP-1, and G-CSF levels in muscle tissues than any other vaccine, but similar serum cytokine profiles were observed to those induced by the other vaccines. Currently available vaccines did not induce muscular degeneration or fibrotic scar as observed with muscle contracture caused by multiple IMs of antibiotics in the past.

The pathogenesis of acute lung injury induced by hemorrhagic shock

Background Acute lung injury (ALI) is an important cause for mortality and morbidity of patients with traumatic hemorrhagic shock.Objective To provide a review about the possible pathogenesis of ALI induced by traumatic hemorrhagic shock.Content Activity and interaction of polymorph nuclear neutrophils(PMNs),Toll-like receptors(TLRs),hypoxia-inducibic factor(HIF) and nuclear factor-κB(NF-κB) are involved in the pathogenesis of ALI induced by traumatic hemorrhagic shocks,and play important roles.Trend To study the pathogenesis of ALI induced by traumatic hemorrhagic shock and the interaction of injury factors will contribute to the treatment of this disorder. Key words: Acute lung injury; Trauma; Hemorrhagic shock; Toll-like receptors; Hypoxia-inducibic factor

Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro

BackgroundBacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection.MethodsUsing an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot.ResultsPMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection.ConclusionsOur findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

Short term effects of alpha1-antitrypsin substitution therapy on the degranulation of neutrophil granulocytes

Introduction: Alpha-1-antitrypsin-deficiency (AATD) is a hereditary, co-dominant condition which results in low levels of circulating alpha-1-antitrypsin (A1AT), unbalanced protease activity and frequent development of emphysema. The concept behind substitution therapy with human plasma A1AT is to normalize the concentration in blood and tissue and thereby protecting the lungs from progressive destruction by proteases.

Transcellular migration of neutrophil granulocytes through the blood-cerebrospinal fluid barrier after infection with Streptococcus suis

BackgroundA critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions.MethodsIn an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1.ResultsHere, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome®-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation.ConclusionOur data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.

Influences of treatment of early pregnant mares with the progestin altrenogest on embryonic development and gene expression in the endometrium and conceptus

A positive influence of altrenogest treatment on a retarded development of the conceptus around the beginning of placentation in mares older than 8 years could be recently demonstrated. In the present study, effects of altrenogest treatment in early-pregnant mares on conceptus development and expression of endometrial and embryonic genes were investigated. Genes were chosen according to a possible involvement in embryo-maternal interaction and embryonic development in the equine species. Mares were treated with altrenogest (0.044 mg/kg bodyweight) or sunflower oil (placebo) from day 5 to 11 after ovulation. Embryos (altrenogest n = 13, placebo n = 12) and biopsies were collected on day 11. Pregnancy rate and embryonic size were not influenced by treatment (embryonic diameter: altrenogest 7.0 ± 2.5, placebo 6.5 ± 1.7 mm, n.s.). The percentage of luminal epithelial cells, superficial glandular epithelial cells and interstitial cells with nuclei staining positively for the progesterone receptor was significantly lower (P < 0.05) in samples collected from altrenogest-treated than from placebo-treated mares (e.g., luminal epithelium: altrenogest 1.9 ± 1.7%, placebo 23.0 ± 10.5%, P < 0.05). Staining for COX2 (cyclooxygenase-2) was not affected by treatment. In the endometrium a slight but significant increase in the number of PMN (polymorph nuclear neutrophils) was seen in response to treatment (altrenogest 0.8 ± 0.5 PMN/field, placebo 0.3 ± 0.3 PMN/field; P < 0.05). No differences in the relative gene expression of COX2, the receptors for progesterone, estrogens and growth hormone as well as for IGF (insulin-like growth factor) 1 and 2 were detected. The relative gene expression of aquaporin 3 in relation to β-actin differed significantly (P < 0.05) between embryos from altrenogest (3.2 ± 0.8) and placebo-treated mares (1.3 ± 0.2), but no other genes were affected. The study demonstrates down-regulation of progesterone receptors in the endometrium of early pregnant mares by treatment with the progestin altrenogest. This increased expression of aquaporin 3 in the conceptus was not related to changes in embryonic size or development.

Chlamydia pneumoniae Hides inside Apoptotic Neutrophils to Silently Infect and Propagate in Macrophages

BackgroundIntracellular pathogens have developed elaborate strategies for silent infection of preferred host cells. Chlamydia pneumoniae is a common pathogen in acute infections of the respiratory tract (e.g. pneumonia) and associated with chronic lung sequelae in adults and children. Within the lung, alveolar macrophages and polymorph nuclear neutrophils (PMN) are the first line of defense against bacteria, but also preferred host phagocytes of chlamydiae.Methodology/Principal FindingsWe could show that C. pneumoniae easily infect and hide inside neutrophil granulocytes until these cells become apoptotic and are subsequently taken up by macrophages. C. pneumoniae infection of macrophages via apoptotic PMN results in enhanced replicative activity of chlamydiae when compared to direct infection of macrophages, which results in persistence of the pathogen. Inhibition of the apoptotic recognition of C. pneumoniae infected PMN using PS- masking Annexin A5 significantly lowered the transmission of chlamydial infection to macrophages. Transfer of apoptotic C. pneumoniae infected PMN to macrophages resulted in an increased TGF-ß production, whereas direct infection of macrophages with chlamydiae was characterized by an enhanced TNF-α response.Conclusions/SignificanceTaken together, our data suggest that C. pneumoniae uses neutrophil granulocytes to be silently taken up by long-lived macrophages, which allows for efficient propagation and immune protection within the human host.

Incompetence of Neutrophils to Invasive Group A streptococcus Is Attributed to Induction of Plural Virulence Factors by Dysfunction of a Regulator

Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection.

Embryo transfer induces a subclinical endometritis in recipient mares which can be prevented by treatment with non-steroid anti-inflammatory drugs

We tested the hypothesis that subclinical endometritis occurs after embryo transfer (ET) in the horse. Recipient mares were treated with meclofenamic acid (M) or flunixin meglumin (F) after ET or were left untreated ( n = 9 per group). Embryos were re-collected 4 days after transfer. Endometrial biopsies were taken for histology and analysis of cyclooxygenase-2 (COX-2) by immunohistochemistry and for PCR. Bacteriological swabs were collected from the uterus and lavage fluid of donor and recipient mares. Progesterone and prostaglandin F 2α release was analysed in recipient mares after ET. Four days after ET, four embryos were recovered from group M and three from group F and untreated mares, each. The number of polymorph nuclear neutrophils was reduced in treated mares ( p < 0.05). Expression of mRNA for inflammatory cytokines did not differ between groups. In group M, expression of endometrial prostaglandin-E-synthase was higher than in group F ( p < 0.05). Three out of nine control mares unterwent preterm luteolysis ( p < 0.05 vs. treatment groups), prostaglandin release ( p < 0.05) and the number of COX-2 positive cells ( p < 0.01) were significantly higher than in treated mares. Only few bacteriological swabs were positive. In conclusion, treatment of embryo recipient mares with non-steroid anti-inflammatory drugs inhibits the inflammatory response of the endometrium after ET. Meclofenamic acid may have advantages in comparison to flunixin meglumin due to a different influence on prostaglandin synthesis that may not result in inhibition of embryonic mobility.

Herpes virus entry mediator synergizes with Toll‐like receptor mediated neutrophil inflammatory responses

In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the function of HVEM on PMN, we evaluated the effect of receptor ligation on human PMN effector functions using an agonistic monoclonal antibody. Here we demonstrate that activation of HVEM causes activation of neutrophil effector functions, including respiratory burst, degranulation and release of interleukin-8 in synergy with ligands for Toll-like receptors or GM-CSF. In addition, stimulation via HVEM enhanced neutrophil phagocytic activity of complement opsonized, but not of non-opsonized, particles. In conclusion, these results indicate a new, as yet unknown, participation of HVEM in the innate immune response and points to a new link between innate and adaptive immunity.