Polymorphonuclear Neutrophil Granulocytes Research Articles

Ethanol‐Induced Malfunction of Neutrophils Respiratory Burst on Patients Suffering From Alcohol Dependence

Polymorphonuclear, neutrophil granulocytes (PMN) play a major role in the control of infections, and people who abuse alcohol are susceptible to infections. Resistance against infections ensues intracellularly following initial phagocytosis of microorganisms with the oxygen-dependent respiratory burst, the key enzyme of which is the respiratory burst oxidase, whereby oxygen radicals are produced for microbial destruction. To date there is insufficient information available in connection with the process of impaired defence against infection in patients suffering from alcohol dependence. Therefore, our investigation was carried out to determine the influence of alcohol exposition on the formation of oxygen radicals and the respiratory burst. 4.5 ml of whole blood was taken from 10 healthy adults and 10 patients suffering from alcohol dependence. An additional 3.5 ml of whole blood was taken from the alcoholic patients for determination of the blood alcohol concentration. The respiratory burst of PMN was tested using the Four-Colour-Continuous Flow Cytometer. Each experimental procedure consisted of 4 test samples [negative controls, Escherichia coli, FMLP-supplement (N-formyl-l-methionyl-l-leucyl-l-phenylalanin), PMA-supplement (phorbol-12-myristate-13-acetate)]. Differing concentrations of ethanol were also introduced to each of the tests performed (0.20 to 4.00 g/l). Ethanol revealed a marked decrease of burst activity in those patients suffering from alcoholism with increased alcohol concentration. A dependence between the burst activity and the ethanol concentration was seen to be statistically significant. This effect was only evident after stimulation with E. coli and FMLP in those patients with alcohol dependence. The results presented in this study show an impairment in the function of PMN in those patients addicted to alcohol due to the decrease in burst activity. In view of the results of the different stimuli, the second-messenger effects were not evident. A clarification of this phenomenon could well be assumed as an allosteric receptor effect on the burst oxidase, namely, a direct effect on the phagocytosis interaction between circulating granulocytes and causative organisms.

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Myeloperoxidase Delays Neutrophil Apoptosis Through CD11b/CD18 Integrins and Prolongs Inflammation

Polymorphonuclear neutrophil granulocytes have a central role in innate immunity and their programmed cell death and removal are critical for efficient resolution of acute inflammation. Myeloperoxidase (MPO), a heme protein abundantly expressed in neutrophils, is generally associated with killing of bacteria and oxidative tissue injury. Because MPO also binds to neutrophils, we investigated whether MPO could affect the lifespan of neutrophils. Here, we report that MPO independent of its catalytic activity through signaling via the adhesion molecule CD11b/CD18 rescued human neutrophils from constitutive apoptosis and prolonged their life span. MPO evoked a transient concurrent activation of extracellular signal-regulated kinase and Akt, leading to phosphorylation of Bad at both Ser112 and Ser136, prevention of mitochondrial dysfunction, and subsequent activation of caspase-3. Consistently, pharmacological inhibition of extracellular signal-regulated kinase, Akt, or caspase-3 reversed the antiapoptosis action of MPO. Acute increases in plasma MPO delayed murine neutrophil apoptosis assayed ex vivo. In a mouse model of self-resolving inflammation, MPO also prolonged the duration of carrageenan-induced acute lung injury, as evidenced by enhanced alveolar permeability and accumulation of neutrophils parallel with suppression of neutrophil apoptosis. Our results indicate that MPO functions as a survival signal for neutrophils and thereby contribute to prolongation of inflammation.

Automation and validation of a rapid method to assess neutrophil and monocyte activation by routine fluorescence flow cytometry in vitro

The aim of the present study was to design an automated-gating hematology fluorescence flow cytometry methodology permitting the assessment of neutrophil and monocyte activation in EDTA-anticoagulated whole blood based on cell granularity, lipid membrane components, cell shape and volume, and total cell nucleic acid (NA) compounds. For particularly monitoring the proper functioning of patients' innate immune system as the first line defense against microbial invaders, the suitable test system should be rapid, simple, reliable by yielding reproducible results. It must be validated against established methods, and it must prove to work in selected clinical settings, e.g. in intensive care unit (ICU) environments. The adaptation of a routine hematology cell analyser utilizing fluorescence flow cytometry resulted in a potentially useful system for all requirements. It proved to detect in real-time and in a reliable and reproducible way the main cellular response reactions of neutrophils and monocytes during externally stimulated immune defense. Validation was successful when comparing it to established methods. The quantified activation effects were dose dependent from the applied activating agents. Cellular response kinetics could be measured and described and showed to be in line with the prevailing cell response models. Upon applying the test method to a healthy population of volunteers and a first cohort of ICU patients with and without evident immune depression, the test revealed excellent cellular responses to external activating cytotoxic stimuli (lipopolysaccharide; LPS) for the control group, slightly weaker response from ICU patients without immune depression and no response from patients with evident immune depression.We conclude that routine hematology fluorescence flow cytometry can accurately and reproducibly measure different activation steps of monocytes and polymorphonuclear neutrophilic granulocytes to defined external stimuli. This may potentially be applied as a STAT (Latin statim = immediately) and routine screening and surveillance method for inflammatory diseases.

The secretory leukocyte protease inhibitor (SLPI) and the secondary granule protein lactoferrin are synthesized in myelocytes, colocalize in subcellular fractions of neutrophils, and are coreleased by activated neutrophils

The secretory leukocyte protease inhibitor (SLPI) re-establishes homeostasis at sites of infection by virtue of its ability to exert antimicrobial activity, to suppress LPS-induced cellular immune responses, and to reduce tissue damage through inhibition of serine proteases released by polymorphonuclear neutrophil granulocytes (PMNs). Microarray analysis of bone marrow (BM) populations highly enriched in promyelocytes, myelocytes/metamyelocytes (MYs), and BM neutrophils demonstrates a transient, high mRNA expression of SLPI and genuine secondary granule proteins (GPs) in MYs. Consistent with this finding, immunostaining of BM cells showed SLPI and the secondary GP lactoferrin (LF) to be present in cells from the myelocyte stage and throughout neutrophil differentiation. Subcellular fractionation studies demonstrated the colocalization of SLPI and LF in subcellular fractions highly enriched in secondary granules. Finally, exocytosis studies demonstrated a corelease of SLPI and LF within minutes of activation. Collectively, these findings strongly indicate that SLPI is localized in secondary granules of PMNs. However, the amount of SLPI detected in PMNs is low compared with primary keratinocytes stimulated by growth factors involved in wound healing. This implicates that neutrophil-derived SLPI might not contribute essentially to re-establishment of homeostasis at sites of infection but rather, exert physiologically relevant intracellular activities. These might include the protection of secondary GPs against proteolytic activation and/or degradation by proteases, which might be dislocated to secondary granules at minute amounts as a consequence of spillover.

Комплексне морфометричне дослідження поліморфноядерних нейтрофільних гранулоцитів крові при експериментальному цукровому діабеті

The results of investigation of morphological peculiarities of neutrophils in blood of rats with experimental diabetes mellitus (EDM) caused by L-arginine and aminoguanidine are presented. Neutrophils’ size under EDM was from 10 to 14 µm, but their size decreased to 9 µm after the administration of L-arginine and aminoguanidine. It should be noted that under EDM the regulatory properties and reserve potential of the cytoplasmatic membranes of neutrophils displayed positive effect caused by L-arginine and aminoguanidine. Substantial inconsistency in peripheral blood neutrophil size in rats with streptozotocin diabetes was established. These parameters may serve as objective diagnostic criterions of this disease and as prognostically important marker during treatment of this pathological state. Keywords: polymorphonuclear neutrophil granulocytes, experimental diabetes mellitus, L-arginine, aminoguanidine.

Influence of fluoroquinolones on phagocytosis and killing ofCandida albicansby human polymorphonuclear neutrophils

Candida albicans infections often occur during or shortly after antibacterial treatment. Phagocytosis by polymorphonuclear neutrophil granulocytes (PMN) is the most important primarily defence mechanism against C. albicans. Certain antibiotics such as some fluoroquinolones (FQ) are known to influence phagocyte functions. Thus, we investigated the influence of older and newer FQ on the phagocytosis and killing of C. albicans by human PMN paying special attention to CD11b expression of these cells as an indicator of the degree of their activation. In order to obtain comprehensive and comparable results we tested 13 FQ over a wide range of concentrations and in a time dependent manner in a standardized approach. When used at therapeutic concentrations, the FQ tested did not influence to a clinically significant degree the phagocytosis or the killing of C. albicans by human PMN and also not their activation. However, at high concentrations those FQ with cyclopropyl-moiety at position N1 showed increase in CD11b expression and diminished phagocytosis and oxidative burst.

Comparison of Three Common Methods of Lung Lavage in Healthy Pigs

Bronchoscopic, endotracheal and transtracheal lung lavage were evaluated in 38 healthy pigs taken from a nucleus herd in a good state of health with respect to their applicability in practice and the traceability of bacteria, cellular parameters and the antimicrobial peptide PR-39 in the respective lavage fluid samples. The total cell count, qualitative morphological cellular characteristics as well as PR-39 could be determined in all lavage fluid samples, while quantitative cell differentiation was not possible in endotracheal lavage samples. The comparison of the three methods resulted in a higher proportion of polymorphonuclear neutrophil granulocytes (PMNs) and higher concentrations of PR-39 in transtracheal samples. For this reason different valuation standards with respect to PMNs and PR-39 concentrations are presupposed for transtracheal lavage samples. The occurrence of pavement epithelial cells as well as the number of contaminating bacterial species per sample was the lowest in transtracheal lavage. Mycoplasma hyopneumoniae polymerase chain reaction appeared to have the highest diagnostic sensitivity in combination with bronchoscopic lavage. In conclusion, bronchoscopic and transtracheal lavage were considered to be more appropriate for bacteriological and cytological diagnostics than endotracheal lavage.

Apoptosis driven infection

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic “eat me” signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic “eat me” signal PS to establish infection.

The Potential of BORIS Detected in the Leukocytes of Breast Cancer Patients as an Early Marker of Tumorigenesis

Brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer-testis antigen gene family. These genes are normally expressed only in spermatocytes but abnormally activated in different malignancies, including breast cancer. The aim of this study was to investigate the expression of BORIS in the leukocytes of breast cancer patients and the correlation between BORIS levels and clinical/pathologic variables. Leukocytes were obtained from whole blood of 87 breast cancer patients and 52 donors not diagnosed with cancer. BORIS protein was detected in leukocytes by immunohistochemical staining; the immunoreactivity score (IRS) of each sample was determined. Additionally, BORIS expression was assessed by Western blot analysis and real-time reverse transcription-PCR. We describe significantly high levels of BORIS (IRS = 4.25 +/- 0.034) in a subpopulation of leukocytes, the neutrophil polymorphonuclear granulocytes, in 88.5% of breast cancer patients. Increased IRS for BORIS in these patients correlated with increased tumor size. In comparison, 19.2% samples from the control group were BORIS positive with only very low levels of BORIS (IRS = 0.25 +/- 0.009). We report here the novel finding of BORIS expression in polymorphonuclear granulocytes of breast cancer patients. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. We conclude that BORIS represents a new class of cancer biomarkers different from those currently used in medical practice.

Neutrophil Granulocytes Are Required for EffectiveBacillus Calmette-GuérinImmunotherapy of Bladder Cancer and Orchestrate Local Immune Responses

The role of polymorphonuclear neutrophil granulocytes (PMN) in antitumoral immune responses displays a striking dichotomy. Under inflammatory conditions, PMN may promote tumor growth and progression. In contrast, especially in the context of therapeutic interventions, PMN can exert important antitumor functions. However, until now, the mechanisms of PMN-mediated activation of tumor immunity are poorly defined. Based on a murine model of Bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer, we provide evidence for a novel immunoregulatory role of this leukocyte subset. PMN immigrate into the bladder after intravesical BCG instillation and depletion of PMN from tumor-bearing mice completely abrogated antitumor efficacy of BCG. PMN stimulated with BCG in vitro as well as PMN isolated from the urine of BCG-treated patients were a major source of the chemokines interleukin-8, growth-related oncogene-alpha, macrophage inflammatory protein-1 alpha and of the inflammatory cytokine migration inhibitory factor. In vitro, BCG-stimulated PMN indirectly induced T-cell chemotaxis via the accessory function of activated monocytes. In vivo, depletion of PMN from BCG-treated mice significantly impaired CD4(+) T-cell trafficking to the bladder. These data show that PMN direct the migration of effector cells to the bladder and by this means are indispensable for effective tumor immunotherapy. Thus, our findings provide evidence for a novel early immunoregulatory role of these innate immune cells in local antitumor immunity.

Impact of carbon dioxide versus air pneumoperitoneum on peritoneal cell migration and cell fate

Postoperative systemic immune function is suppressed after open abdominal surgery, as compared with that after minimally invasive abdominal surgery. As a first line of defense, peritoneal macrophages (PMo) and polymorphonuclear neutrophil granulocytes (PMNs) are of primary importance in protecting the body from microorganisms. Previous studies have shown changes in these cell populations over time after open versus laparoscopic surgery. This study aimed to investigate the dynamics of cell recruitment and clearance of peritoneal cells. Female NMRI mice (33 +/- 2 g) were randomly assigned to carbon dioxide (CO2) or air insufflation. Intravasal cells with phagocytic capabilities were selectively stained by intravenous injection of the fluorescent dye PKH26 24 h before surgery. Gas was insufflated into the peritoneal cavity through a catheter, and the pneumoperitoneum was maintained for 30 min. Peritoneal lavage was performed 1, 3, 8, or 24 h after surgery. Apoptotic cells were assessed by flow cytometry using a general caspase substrate. The total peritoneal cell count did not differ between groups. The PKH26-positive PMo level was significantly increased after CO2, as compared with air, at 1 h and 24 h. The ratio of apoptotic PMo did not differ between the groups. In the peritoneal lavage, polymorphonuclear leukocytes (PMNs) were tripled in the air group, as compared with the CO2 group, whereas the ratio of apoptotic PMNs was significantly decreased. There was a higher fraction of PKH26-positive PMNs after air exposure, as compared with that after CO2. Air exposure triggered a higher transmigration rate of PMNs from the blood compartment into the peritoneal cavity and decreased PMN apoptosis, as compared with CO2. The lower proportion of PKH26-positive peritoneal macrophages in the air group might have been attributable to a higher inflammatory stimulation than in the CO2 group, leading to increased emigration of PMo to draining lymph nodes. All the findings underscore a complex cell-specific regulation of cell recruitment and clearance in the peritoneal compartment.

Ultrasound exposure can increase the membrane permeability of human neutrophil granulocytes containing microbubbles without causing complete cell destruction

Activated polymorphonuclear neutrophil (PMN) granulocytes can bind and subsequently phagocytose microbubbles used as ultrasound (US) contrast agents. The purpose of the present study was to assess insonation effects on cell membrane integrity and metabolic activity of activated PMN. Furthermore, we investigated whether or not there is an acoustic threshold at which insonation of PMN results in increase of membrane permeability without causing complete cell destruction. PMN isolated from healthy volunteers were activated with phorbol myristate acetate (PMA) for 15 min to allow phagocytosis of albumin and lipid microbubbles and were subsequently exposed to US with a mechanical index between 0.15 and 1.8. Apoptosis, loss of membrane integrity and formation of cell fragments were evaluated by measurement of lactate dehydrogenase leakage and by double staining with annexin V and propidium iodide, using flow cytometry. Neutrophil superoxide anion generation was measured photometrically. Insonation of activated PMN in the presence of microbubbles amplified apoptosis and lactate dehydrogenase leakage and induced loss of membrane integrity and complete cell destruction with increasing acoustic pressures. The bioeffects observed by insonation with high mechanical indices (1.0 to 1.8), and particularly the formation of cell fragments, were significantly more pronounced in the presence of albumin microbubbles. Insonation in the presence of lipid microbubbles increased cell membrane permeability, but caused significantly less cell destruction and left the metabolic activity of activated PMN uninfluenced. Thus, both albumin and lipid microbubbles induce apoptosis and membrane injury during insonation of activated PMN. However, insonation in the presence of lipid microbubbles seems to influence cell viability to a smaller extent. This could be of advantage in the setting of US-guided local drug delivery. In this setting, increase of membrane permeability may allow bioactive substances to enter into cells, which survive the US treatment, and specifically modify their function. (E-mail: Grigorios_Korosoglou@med.uni-heidelberg.de)

Fate of intraperitoneally injected fluorescent microspheres in developing Ictalurus punctatus

Fluorescent microspheres (FMS) were injected intraperitoneally into channel catfish fry at 2 days post hatch (dph), 1, 2, 3, 4 and 8 weeks post hatch (wph). The FMS were observed in the vasculature almost immediately after injection in all age groups except 2 dph. Fluorescent microspheres were observed within mononuclear phagocytes in the vasculature after 0.16 dph in all age groups. Fluorescent microspheres were first phagocytized in the coelomic cavity immediately after injection, while the majority of coelomic FMS were phagocytized between 0.16 and 1 dph for all ages. Enzyme cytochemical staining indicated that both polymorphonuclear (neutrophilic granulocytes) and mononuclear phagocytes had phagocytized FMS in the coelomic cavity and organs, with a predominance of FMS found in mononuclear phagocytic cells in all age groups across all sample periods. The predominant organs associated with the observed cellular responses were the posterior kidney, spleen, and anterior kidney. Splenic organization and melanomacrophage development and activity were more pronounced as the fish aged from 2 wph on. Particulate clearance rates were faster in the 2 dph and 1 wph fish than the older ages of fish. These results suggest that to facilitate particulate retention, channel catfish should be vaccinated at 4 wph or older.

Rapid leukocyte activation following intraarticular bleeding

The study aims at elucidating the leukocyte activation in the joint fluid of patients with acute traumatic hemarthrosis. Paired samples of peripheral blood and articular effusions after an acute hemorrhage were obtained from 22 patients. Leukocytes were separated and stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD11a, CD18, and CD97 monoclonal antibodies for flow cytometry. The reactive oxygen species (ROS) production of leukocytes in corresponding samples of peripheral blood and joint effusion was measured via luminol dependent whole blood chemiluminometry. Significant decrease of CD11a density on monocytes, but markedly enhanced expression of CD97 and CD18 on polymorphonuclear neutrophil granulocytes (PMN), and significantly increased proportion of CD97 positive lymphocytes were found in the joint fluids as compared to the corresponding peripheral blood samples. Moreover, significantly decreased lag time and elevated rate of ROS production were revealed by chemiluminometry in case of joint derived leukocytes. Our results provide good evidence for intraarticular leukocyte activation during acute hemarthrosis. Since the activation precedes synovial inflammation, it is suggested that the leukocytes play an important role as an initiator in the pathogenesis of acute hemarthrosis.

Massive blood transfusion induces characteristic monocyte messenger RNA expression patterns in patients after multiple major trauma: a microarray pilot study

Objective: Polymorphonuclear neutrophil (PMN) granulocytes play an important role in trauma, shock, and sepsis. In these acute illness situations, multiple parameters can be used to describe functional and structural changes of PMN granulocytes like, for example, cell count, multiple surface adhesion molecules, free radical oxygen production, activity of phagocytosis, and cytokine production. Nevertheless, up to now, in most of the daily clinical situations, only the general white blood cell count is used to receive information, ignoring the fact that the leukocyte-cell system is a mix of different complex cell-subsystems with totally different functions. Considering that complex systems show nonlinear behavior, we could expect heterogeneous reactions of PMN granulocytes to near-identical stimulations. Therefore, functional and structural parameters of this cell type were assessed during an ischemiareperfusion situation due to an identical extremity trauma impact. Material and Methods: Twenty male patients; elective anterior cruciate ligament reconstruction with tourniquet-induced ischemia; structural parameters: CD11b, CD16, CD18, CD62L, CD55, CD63, CD95; functional parameters: cell count, respiratory burst (free radical oxygen production) with and without stimulation by tumor necrosis factor, PMA, or fMLP. Analysis by fluorescence cytometry. Results: Each parameter showed uniformed changes in all patients. This could be interpreted as a homogenous reaction to homogenous stimulations. Nevertheless, in the analysis of all 20 patients intraindividually, it can be demonstrated that summarizing all functional and structural parameters each patient showed a total different pattern of PMN granulocyte reactions to a homogenous ischemia-reperfusion trauma. Conclusions: To describe cell behavior in acute illness situations, it is not adequate to use the routine, for example, white blood cell count. Even the analysis of single bhigh-techQ parameters can describe the principle change of the complex cell system only. It is necessary to analyze the pattern of cell behavior intraindividually to get deeper insights for a better understanding of reactions to, for example, trauma situations.

Dexamethasone depresses the expression of l-selectin but not the in vivo migration of bovine neutrophils into the uterus

There are several indications that periparturient depression of functional properties of polymorphonuclear neutrophilic granulocytes (PMN) may be of great importance for the pathogenesis of genital infections after calving. As the periparturient period is characterized by high plasma levels of corticosteroids, the hypothesis to be tested was that high concentrations of glucocorticoids during the periparturient period suppress uterine PMN in number and functionality. An in vivo endometritis model was applied to examine uterine PMN. Recombinant human interleukin-8 (rhIL-8) was infused into the uterus of estrous cows and heifers 24 h after pretreatment with dexamethasone (0.07 mg/kg i.m.). Six hours after rhIL-8 infusion high numbers of uterine PMN were isolated and characterized as to their immunophenotype and function. Animals treated with dexamethasone animals showed leucocytosis due to neutrophilia in peripheral blood. Despite a downregulation of expressed l-selectin, cattle treated with dexamethasone showed more uterine PMN than those treated with placebo. Dexamethasone decreased plasma concentrations of the immunomodulatory steroidal hormones cortisol and estrogen. Dexamethasone directly reduced the generation of reactive oxygen species (ROS) by uterine PMN. This may be a useful mechanism since it would protect the endometrium from tissue damage by excessive extracellular ROS. However, it is not known if the net effect is in fact a reduction in ROS, as the number of uterine cells increases. Our study shows that glucocorticoids may not be considered immunosuppressive in all cases and may play an important role in the regulation of post partum uterine defense mechanisms.

Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.

Morphological characterization and in vitro biocompatibility of a porous nickel–titanium alloy

Disks consisting of macroporous nickel–titanium alloy (NiTi, Nitinol ®, Actipore ®) are used as implants in clinical surgery, e.g. for fixation of spinal dysfunctions. The morphological properties were studied by scanning electron microscopy (SEM) and by synchrotron radiation-based microtomography (SRμCT). The composition was studied by X-ray diffractometry (XRD), differential scanning calorimetry (DSC), and energy-dispersive X-ray spectroscopy (EDX). The mechanical properties were studied with temperature-dependent dynamical mechanical analysis (DMA). Studies on the biocompatibility were performed by co-incubation of porous NiTi samples with isolated peripheral blood leukocyte fractions (polymorphonuclear neutrophil granulocytes, PMN; peripheral blood mononuclear leukocytes, PBMC) in comparison with control cultures without NiTi samples. The cell adherence to the NiTi surface was analyzed by fluorescence microscopy and scanning electron microscopy. The activation of adherent leukocytes was analyzed by measurement of the released cytokines using enzyme-linked immunosorbent assay (ELISA). The cytokine response of PMN (analyzed by the release of IL-1ra and IL-8) was not significantly different between cell cultures with or without NiTi. There was a significant increase in the release of IL-1ra ( p < 0.001 ), IL-6 ( p < 0.05 ), and IL-8 ( p < 0.05 ) from PBMC in the presence of NiTi samples. In contrast, the release of TNF- α by PBMC was not significantly elevated in the presence of NiTi. IL-2 was released from PBMC only in the range of the lower detection limit in all cell cultures. The material, clearly macroporous with an interconnecting porosity, consists of NiTi (martensite; monoclinic, and austenite; cubic) with small impurities of NiTi 2 and possibly NiC x . The material is not superelastic upon manual compression and shows a good biocompatibility.