Polymorphonuclear Neutrophil Leukocytes Research Articles

Hematopoietic Interferon Regulatory Factor 8-Deficiency Accelerates Atherosclerosis in Mice

Inflammatory leukocyte accumulation drives atherosclerosis. Although monocytes/macrophages and polymorphonuclear neutrophilic leukocytes (PMN) contribute to lesion formation, sequelae of myeloproliferative disease remain to be elucidated. We used mice deficient in interferon regulatory factor 8 (IRF8(-/-)) in hematopoietic cells that develop a chronic myelogenous leukemia-like phenotype. Apolipoprotein E-deficient mice reconstituted with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow displayed an exacerbated atherosclerotic lesion formation compared with controls. The chronic myelogenous leukemia-like phenotype in mice with IRF8(-/-) bone marrow, reflected by an expansion of PMN in the circulation, was associated with an increased lesional accumulation and apoptosis of PMN, and enlarged necrotic cores. IRF8(-/-) compared with IRF8(+/+) PMN displayed unaffected reactive oxygen species formation and discharge of PMN granule components. In contrast, accumulating in equal numbers at sites of inflammation, IRF8(-/-) macrophages were defective in efferocytosis, lipid uptake, and interleukin-10 cytokine production. Importantly, depletion of PMN in low-density lipoprotein receptor or apolipoprotein E-deficient mice with IRF8(-/-) or IRF8(-/-) apolipoprotein E-deficient bone marrow abrogated increased lesion formation. These findings indicate that a chronic myelogenous leukemia-like phenotype contributes to accelerated atherosclerosis in mice. Among proatherosclerotic effects of other cell types, this, in part, is linked to an expansion of functionally intact PMN.

The influence of different anticoagulants and sample preparation methods on measurement of mCD14 on bovine monocytes and polymorphonuclear neutrophil leukocytes

BackgroundMembrane-CD14 (mCD14) is expressed on the surface of monocytes, macrophages and polymorphonuclear neutrophil leukocytes (PMN). mCD14 acts as a co-receptor along with Toll like receptor 4 (TLR 4) and MD-2 for the detection of lipopolysaccharide (LPS). However, studies using different sample preparation methods and anticoagulants have reported different levels of mCD14 on the surface of monocytes and neutrophils. In this study, the influence of various anticoagulants and processing methods on measurement of mCD14 on monocytes and neutrophils was examined.ResultsWhole blood samples were collected in vacutainer tubes containing either sodium heparin (HEPARIN), ethylenediaminetetraacetic acid (EDTA) or sodium citrate (CITRATE). mCD14 on neutrophils and monocytes in whole blood samples or isolated cells was measured by the method of flow cytometry using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. There was a significant difference (p < 0.05) in the mean channel fluorescence intensity (MFI) of mCD14 on neutrophils in whole blood samples anticoagulated with HEPARIN (MFI = 64.77) in comparison with those in whole blood samples anticoagulated with either EDTA (MFI = 38.25) or CITRATE (MFI = 43.7). The MFI of mCD14 on monocytes in whole blood samples anticoagulted with HEPARIN (MFI = 206.90) was significantly higher than the MFI in whole blood samples anticoagulated with EDTA (MFI = 149.37) but similar to that with CITRATE (MFI = 162.55). There was no significant difference in the percentage of whole blood neutrophils or monocytes expressing mCD14 irrespective of type of anticoagulant used. However, MFI of mCD14 on monocytes was about 3.2-folds (HEPARIN), 3.9-folds (EDTA) or 3.7 folds (CITRATE) higher than those on neutrophils. Furthermore, there was no significant difference in mCD14 levels between unprocessed whole blood monocytes and monocytes in peripheral blood mononuclear cell preparation. Conversely, a highly significant difference was observed in mCD14 between unprocessed whole blood neutrophils and isolated neutrophils (p < 0.05).ConclusionFrom these results, it is suggested that sodium heparin should be the preferred anticoagulant for use in the reliable quantification of the surface expression of mCD14. Furthermore, measurement of mCD14 is best carried out in whole blood samples, both for neutrophils and monocytes.

DNA damage in peripheral blood mononuclear cells and neutrophils of dairy cows during the transition period

This study was designed to investigate the apoptotic process in peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) in dairy cattle during the transition period. Blood samples were collected from 4 dairy cattle at 3 weeks before the expected parturition (wk -3), parturition (wk 0) and 3 weeks after parturition (wk +3). The DNA damage of PBMC and PMN was evaluated based on the comet assay using visual scoring (arbitrary units). Undamaged DNA remained within the core (score 0) and the broken DNA migrated from the core towards the anode forming the tail of a comet (scores 1-4). Significantly higher scores in PBMC at wk 0 and wk +3 were observed compared with those in PMN although there were no significant changes of scores in either cell type during the experimental period. It is suggested that the apoptotic rate of PBMC is accelerated compared with that of PMC during the transition period.

The effects of salbutamol in an experimental model with acute respiratory distress syndrome

ObjectiveTo investigate salbutamol effects on histopathologic features of acute respiratory distress syndrome (ARDS). MethodsARDS was designed in Wistar albino male rats, 250–300 g in weight, by intratracheal instillation of physiological saline solution. Anesthezied and tracheotomized rats with ARDS were pressure-controlled ventilated. At the end of the 210 minutes, two hours past and nebulized salbutamol inhalation was tried. All rats were assigned to two groups: Group 1 (n=10) control group, given no treatment, group 2 (n=10) received salbutamol. Nebulized salbutamol inhalation was given in the dosage of 0, 15 mg/kg/dose. Rats were continued to be on ventilator through the experiment. After the last inhalation, two hours past and their both lungs were excised for histopathological examination. ResultsRat-model ARDS had similar histopathological appearance occuring during the acute phase of the acute respiratory distress syndrome in humans. A statistical difference was seen between control and salbutamol group (P=0.002) for HM. The margination of leukocytes was decreased in salbutamol group. The difference was significant (P<0.042). Hemorrhage and interstitial/intraalveolar edema were much lower in 0.15 mg/dose nebulized salbutamol group than that of control group. There was a significant difference statistically between two groups (P<0.001). ConclusionsInhaled salbutamol therapy for ARDS is may be associated with the improvement of inflamation. Besides known effects of salbutamol, the reducing of infiltration of polymorphonuclear neutrophil leukocytes, interstitial/intraalveolar edema, perivascular and/or intraalveolar hemorrhage and hyaline membrane formation should be emphasized.

Nanocoating of Diazoresin/Polysaccharides Multilayer Boosts Biocompatibility of Nitinol Biomedical Devices

The surface of a biomaterial and a biological environment are key factors determining the biocompatibility of the material. Determining biocompatibility of medical implants is a critical step toward approval and clinical application. In this study, the biocompatibility of a covalently attached nanocoating of alginate(Alg)/diazoresin(DR)/heparin(Hep) multilayer has been evaluated by thrombin inactivation, protein adsorption, leukocyte adhesion, cytotoxicity in vitro and subcutaneous implantation in vivo. It was found that the outermost layer of the coating played a key role on anticoagulant activity. No significant difference was seen in albumin adsorption between uncoated and coated one. However, the fibrinogen adsorption on DR/polysaccharides coatings was lower than the uncoated Nitinol. Compared with the uncoated Nitinol surface, DR/polysaccharides multilayer coating presented a drastically reduced adhesion in vitro of polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC). Cell biocompatibility tests indicated that the nanocoating of DR/polysaccharides multilayer did not lead to an increase in the cytotoxicity of the Nitinol and the adherent cells maintained good viability. Subcutaneous implantation in C57BL/6 mice suggested that the coated Nitinol had good tissue biocompatibility. The use of alginate interlayer for heparin immobilization instead of proteins such as albumin, collagen, and gelatin may avoid the risk of contamination with pathogenic agents. Hence, the nanocoating of covalently attached DR/polysaccharide multilayer boosts the biocompatibility of Nitinol biomedical devices. Keywords: heparin, thrombin, Nitinol, alginate, biocompatibility, Layer-by-layer, Nitinol Biomedical Devices, medical implants, Diazoresin

DNA damage of blood lymphocytes and neutrophils in cattle with lymphosarcoma

&amp;nbsp; The objective of the present study was to analyze the apoptotic process in peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) in cows clinically affected with lymphosarcoma. Thirteen cows were studied. Of them, eight, that were referred because of inappetance, loss of body condition, diarrhoea, constipation, protrusion of third eyelid, and exophthalmia, were seropositive for bovine leukemia virus (BLV) based on a serum enzyme-linked immunosorbent assay. Other animals were apparently healthy and were used as controls. DNA damage of PBMC and PMN was assessed using the Comet assay. The results obtained showed a statistically significant difference in DNA damage between the PBMC and PMN isolated from cows infected with BLV compared to PBMC and PMN isolated from healthy cows. This is the first article to document decreased apoptosis of blood PBMC and PMN in cattle in response to BLV infection using the Comet assay.

On, Around, and Through: Neutrophil-Endothelial Interactions in Innate Immunity

This manuscript will review our current understanding of neutrophilic polymorphonuclear leukocyte (neutrophil) interactions with the endothelium during immune and inflammatory responses, focusing on the molecular mechanisms regulating neutrophil adhesion to and migration through the endothelium in response to infection or tissue injury. This is a complex and dynamic area of research and one that has been the topic of several recent comprehensive reviews to which the interested reader is referred (64, 118, 131). By design, this review will begin with a brief review of some basic aspects of neutrophil biology and endothelial adhesion to provide a foundation. The remainder of the review will focus on selected areas of this complex field, specifically the role of the endothelial glycocalyx in regulating neutrophil adhesion and the mechanisms and consequences of migration of neutrophils between (paracellular) and through (transcellular) endothelial cells during egress from the vasculature.

Flow cytometric differential cell counts in milk for the evaluation of inflammatory reactions in clinically healthy and subclinically infected bovine mammary glands

Somatic cell counts (SCC) are generally used as an indicator of udder health. In Germany, a cutoff value of 100,000 cells/mL is currently used to differentiate between healthy and diseased mammary glands. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed evaluation of the udder health status. The aim of this study was to differentiate immune cells in milk of udder quarters classified as healthy based on SCC values of <100,000 cells/mL. Twenty cows were selected and 65 healthy udder quarters were compared with a control group of 15 diseased udder quarters (SCC>100,000 cells/mL). Cells were isolated from milk of all quarters to measure simultaneously percentages of lymphocytes, macrophages, and polymorphonuclear neutrophilic leukocytes (PMNL) by flow cytometric analysis. The bacteriological status of all 80 quarters was also determined. Differential cell count patterns of milk samples (n=15) with extreme low SCC values of ≤6,250 cells/mL revealed high lymphocyte proportions of up to 88%. Milk cell populations in samples (n=42) with SCC values from >6,250 to ≤25,000 cells/mL were also dominated by lymphocytes, whereas DCC patterns of 6 out of 41 milk samples with SCC values from ≥9,000 to ≤46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL (56–75%). In 13 of 15 milk samples of the diseased udder quarters (SCC >100,000 cells/mL), PMNL were categorically found as dominant cell population with proportions of ≥49%. Macrophages were the second predominant cell population in almost all samples tested in relation to lymphocytes and PMNL. Further analysis of the data demonstrated significant differences of the cellular components between udder quarters infected by major pathogens (e.g., Staphylococcus aureus; n=5) and culture-negative udder quarters (n=56). Even the percentages of immune cells in milk from quarters infected by minor pathogens (e.g., coagulase-negative staphylococci; n=19) differed significantly from those in milk of culture-negative quarters. Our flow cytometric analysis of immune cells in milk of udder quarters classified as healthy by SCC <100,000 cells/mL revealed inflammatory reactions based on DCC.

Sevoflurane Reduces Leukocyte and Platelet Adhesion after Ischemia-Reperfusion by Protecting the Endothelial Glycocalyx

Adhesion of polymorphonuclear neutrophils and platelets to the vessel wall contributes to generating ischemia-reperfusion injury. Endothelial adhesion molecules are harbored within the glycocalyx, which covers every healthy vascular endothelium but is deteriorated by ischemia-reperfusion. Pretreating the heart with volatile anesthetics reduces myocardial infarct size and protects against ischemia-reperfusion injury. The authors analyzed a possible protective effect of sevoflurane on the glycocalyx and implications for postischemic cell adhesion. Isolated guinea pig hearts were perfused with crystalloid buffer and subjected to 20 min of global warm ischemia and 10 min of reperfusion. An intracoronary bolus of 3 x 10(6) polymorphonuclear neutrophilic leukocytes or 1 x 10(9) platelets of human origin was applied after reperfusion, either with or without pretreating with 0.5 or 1 minimal alveolar concentration sevoflurane. The number of sequestered cells was calculated from the difference between coronary input and output. Coronary effluent was collected throughout reperfusion to measure shedding of the glycocalyx. Ischemia-reperfusion induced a significant increase in median (interquartile range) adhesion versus control nonischemic hearts of both leukocytes (38.9 (36.3-42.9) vs. 14.5 (13.1-16.0)%) and platelets (25.0 (22.5-27.1) vs. 9.4 (8.4-10.7)%). Shedding was evidenced by eightfold increases in washout of syndecan-1 and heparan sulfate versus basal. Sevoflurane reduced cell adhesion to near basal at 1 minimal alveolar concentration (leukocytes: 21.2% (19.2-23.9%), platelets: 11.5% (10.4-12.0%). Shedding measurements and electron microscopy demonstrated that sevoflurane-treated hearts retained much of their 200 nm-thick glycocalyx. Sevoflurane reduces glycocalyx shedding in the postischemic coronary bed, maintaining the natural cover for endothelial adhesion molecules and, thus, reducing cell adhesion. This may explain beneficial outcomes linked to clinical use of volatile anesthetics after ischemia-reperfusion.

Glutamate utilization promotes meningococcal survival in vivo through avoidance of the neutrophil oxidative burst

Polymorphonuclear neutrophil leucocytes (PMNs) are a critical part of innate immune defence against bacterial pathogens, and only a limited subset of microbes can escape killing by these phagocytic cells. Here we show that Neisseria meningitidis, a leading cause of septicaemia and meningitis, can avoid killing by PMNs and this is dependent on the ability of the bacterium to acquire L-glutamate through its GltT uptake system. We demonstrate that the uptake of available L-glutamate promotes N. meningitidis evasion of PMN reactive oxygen species produced by the oxidative burst. In the meningococcus, L-glutamate is converted to glutathione, a key molecule for maintaining intracellular redox potential, which protects the bacterium from reactive oxygen species such as hydrogen peroxide. We show that this mechanism contributes to the ability of N. meningitidis to cause bacteraemia, a critical step in the disease process during infections caused by this important human pathogen.

Ex vivo phagocytic overall performance of neutrophilic granulocytes and the relation to plasma insulin-like growth factor-I concentrations in dairy cows during the transition period

Impaired function of polymorphonuclear neutrophilic leukocyte (PMNL) during the peripartal period is a major reason for increased susceptibility of dairy cows to infections in this critical interval. Factors dysregulating PMNL function are widely unknown. Insulin-like growth factor (IGF-I) enhanced PMNL functions in vitro. The objective of this study was to investigate the influence of IGF-I and, additionally, β-hydroxybutyrate and nonesterified fatty acid concentrations on phagocytic activity (PA, percentage of viable PMNL) and phagocytic capacity (PC, mean fluorescence intensity of phagocytic PMNL) assessed by flow cytometry. Antepartum (i.e., wk −3, −2, −1; before calving), plasma concentrations of IGF-I were high (80–110ng/mL) without significant differences between primiparous and pluriparous cows (n=18 and n=41, respectively). Concentrations of IGF-I declined toward the week of calving (wk 1). Postpartum (i.e., wk 2, 3, and 4; after calving), IGF-I remained lower than before parturition, with concentrations higher in primiparous compared with those of pluriparous cows. The PA was constant in primiparous cows throughout the study period. Conversely, PMNL of pluriparous cows had a significantly increased and higher PA in wk 2 and 3 postpartum compared with that of primiparous cows. The PC decreased significantly only in primiparous cows the week of calving, whereas the number of PMNL in primiparous cows exceeded that of pluriparous cows significantly. The phagocytic power (PP, a product of PA by PC), but not the phagocytic overall performance (POP, a product of PA, PC, and PMNL number), differed between primiparous and pluriparous cows in wk 3 postpartum. No significant differences in POP were found, except in wk 4 after calving between the primi- and pluriparous cows. In both groups, POP increased in the week of calving (wk 1). In contrast to β-hydroxybutyrate, which was weakly positive correlated with PA and PP in pluriparous cows in the transition period (wk −3 antepartum to wk 4 postpartum), pluriparous animals had weak negative correlations of PMNL number, PA, PP, POP, and IGF-I concentration in this period. In primiparous animals, only PP and PC were weakly negatively correlated with IGF-I in the transition period. Increased plasma IGF-I concentrations were not associated with enhanced phagocytosis function of bovine blood PMNL ex vivo and, thus, can not be regarded as a suitable predictor for this function.

Role of Polymorphonuclear Neutrophils in the Clearance of Enterococcus faecalis Derived from Saliva and Infected Root Canals

Objectives The goal of this study was to measure (1) the ability of polymorphonuclear neutrophil leukocytes (PMNs) to kill oral Enterococcus faecalis strains, (2) up-regulation of inflammatory mediators by PMNs in interaction with E. faecalis, and (3) the ability of E. faecalis to cause inflammation in mouse muscle tissue. Methods Fifteen endodontic and nine saliva strains of E. faecalis were isolated and identified by specific 16S ribosomal RNA (16S rRNA) primers. The bacteria were grown in BHI broth and incubated with mouse PMN in appropriate media to determine the ability of the PMNs to kill the bacteria. In other experiments up-regulation of interleukin (IL)-1α, tumor necrosis factor α (TNF-α), matrix metalloproteinase-8 (MMP-8), and cyclooxygenase (COX)-2 messenger RNA in the PMNs was measured after exposure of the leukocytes to the bacteria using real-time polymerase chain reaction. Finally, the inflammatory potential of and PMN response to E. faecalis suspension in mouse muscle tissue was examined from histological sections using hematoxylin-eosin staining and immunostaining. Results Murine PMNs killed about 80% of the E. faecalis cells in 1 hour, irrespective of the source of isolation of the strains. Quantitative PCR results showed that IL-1α, TNF-α, MMP-8, and COX-2 messenger RNA were markedly up-regulated in E. faecalis–stimulated PMNs or in E. faecalis–invaded muscular tissues. MMP-8 messenger RNA level was positively related to COX-2 messenger RNA level. Histological evaluation and immunostaining disclosed that all E. faecalis strains could recruit PMNs to the local infectious sites and cause abscess formation. Conclusion E. faecalis strains from saliva and infected root canals have the potential to recruit PMNs in the infectious sites leading to inflammation via up-regulation of PMN IL-1α, TNF-α, MMP-8, and COX-2. PMNs can play an important role in killing of E. faecalis.

Immunity to Bacterial Infections

Co-evolution of pathogenic bacteria and hosts has led to the development of an array of virulence genes (the virulome) and a set of mechanisms of defense which constitute the immune system. Mechanisms of innate or non-specific immunity involve mucosal epithelial surface barriers, antibacterial peptides and enzymes, defensive molecules (collectins, complement), and cells (macrophages, dendritic cells, polymorphonuclear neutrophil leukocytes, natural killer cells). The presence of bacterial products in host tissues induces a complex series of non-specific responses which constitute the inflammatory reaction. Inflammation is critical to the resolution of infection, the key process being the ingestion and killing of bacteria by phagocytes, but inflammation also causes tissue damage and contributes to the pathology. The innate immunity is the initial step in the host defense against pathogens, but some pathogenic bacteria with appropriate virulence factors can overcome the innate immunity mechanisms. In these cases, the goal of innate immunity is to contain the infectious process until specific immunity is developed. The specific immune responses are performed by lymphocytes: B cells, T helper (TH) cells, and cytotoxic T lymphocytes (CTL). Specific antibodies are produced by B cells, usually with the cooperation of TH2 cells. In general terms, extracellular bacteria can be killed by antibodies and complement proteins, or cleared by phagocytosis after opsonization by antibodies, and bacterial exotoxins are neutralized by their specific antibodies. In contrast, resistance to intracellular bacteria requires the induction of TH1 and CTL responses. In this review, we will focus on current knowledge about the defensive mechanisms against bacterial infections. We will discuss the innate immunity mechanisms, connections between innate and specific immunity, the key role of T helper 1 (TH1) and TH2 cells, and the antibody and cell-mediated responses. Keywords: Innate immunity, specific immunity, pathogen-associated molecular patterns, pattern recognition receptors, Tolllike receptors, complement, antibodies, cell-mediated immunity, Immunity, Bacterial Infections, pathogenic bacteria, immune system, antibacterial peptides, enzymes, collectins, macrophages, dendritic cells, polymorphonuclear neutrophil leukocytes, natural killer cells, Inflammation, phagocytes, lymphocytes, B cells, T helper (TH) cells, cytotoxic T lymphocytes, TH2 cells, bacterial exotoxins, eukaryotes, endosymbionts, metazoans, chromosomal DNA, bacterio-phage DNA, plasmids, transposons, neutropenia, Lysozyme, Gram-positive bacteria, Gram-negative bacteria, LL-37, cathelicidin, Ficolins, Pentraxins, N-acetyl-glucosamine, C-reactive protein, MBL-associated serine proteases, Legionella pneumophila, Typhimurium, immunoglobulin, Superantigens, N. gonorrhoeae, Chlamydia trachomatis

E0047 Alphalinonenic acid inhibits high glucosemediated endothelial neutrophil adhesion by decreasing adhesion molecule expression via PI3KAkt pathway

ObjectiveNeutrophil-endothelial adhesion is crucial to vascular injury, the major cause of diabetic vascular complications. We studied the mechanism of cardio-protective effect of Alpha-linolenic acid (ALA).MethodsHuman umbilical vein endothelial cells (HUVECs)...

Effects of in vitro insulin and 2,4-thiazolidinedione on the function of neutrophils harvested from blood of cows in different physiological states

Neutrophils [polymorphonuclear neutrophilic leukocytes (PMNL)] were isolated from 26 Holstein cows in different physiological states (12±1.7 d prepartum, n=8; 7±0 d postpartum, n=9; 253±25.2 d postpartum, n=9) and incubated in vitro for 120min in a factorial arrangement of treatments with 0, 1.5, or 15 ng/mL of bovine insulin and 0 or 300 μg/mL of the peroxisome proliferator-activated receptor-γ ligand 2,4-thiazolidinedione (TZD). Following the incubations, PMNL functional assays were performed to determine treatment effects on proxies for total, extracellular, and intracellular generation of reactive oxygen species (ROS), neutrophil extracellular trap formation, and phagocytic killing abilities. The ROS production of PMNL collected from cows at 7 d postpartum was reduced compared with that of PMNL from midlactation and prepartum cows, but neutrophil extracellular trap expression was 23 and 36% greater in PMNL from prepartum cows compared with that in PMNL from midlactation and postpartum cows, respectively. Insulin had no effect on PMNL functional assay results. In contrast, TZD inhibited a measurement of total ROS production by 89%, increased extracellular superoxide generation by 43%, but had no effect on the intracellular ROS measured. Interestingly, TZD did not alter the ability of the PMNL to release neutrophil extracellular traps and engulf or kill Staphylococcus aureus. These findings suggest a possible anti-inflammatory effect of TZD that may result in reduced extracellular oxidative damage with maintenance of PMNL antimicrobial activity.

Histoplasmosis: cytodiagnosis and review of literature with special emphasis on differential diagnosis on cytomorphology

Human infection with Histoplasma capsulatum runs the gamut from asymptomatic to disseminated disease. In immunocompromised patients, a tiny inoculum can lead to widespread disseminated infection. Early diagnosis and initiation of treatment is therefore important. To review the cases of histoplasmosis diagnosed on fine needle aspiration cytology (FNAC) and to discuss the clinical presentation, associated inflammatory response, load of organisms and differential diagnosis on cytomorphology in these cases. Retrospective review of seven cases of histoplasmosis at a tertiary-care centre during the period from 1998 to 2009 was performed. Clinical presentation along with cytomorphological features were studied and discussed in detail. The mean age of patients was 48.6 years and six out of seven were male. History of immunodeficiency (HIV) was available in five cases. Six patients presented with peripheral and/or abdominal lymphadenopathy. One patient had nodular shadows in both lungs and two also had skin lesions. On cytological smears, a variable load of uniform round to oval, about 2-4 microm in diameter, budding yeasts were seen intracellularly (within histiocytes) as well as extracellularly. In one case (HIV positive), these organisms were also seen within neutrophil polymorphonuclear leucocytes. In two cases, an inflammatory response in the form of epithelioid cell granulomas along with multinucleated giant cells was seen. FNAC is a reliable tool to recognize infection with H. capsulatum in tissues. This infection can cause a variable inflammatory response, which should be considered while reporting on such cases.

Evaluation of the function of polymorphonuclear neutrophilic leukocytes in healthy dogs given a high dose of methylprednisolone sodium succinate

To evaluate effects of a high dose of methylprednisolone sodium succinate (MPSS) on function of polymorphonuclear neutrophilic leukocytes (PMNs) in dogs. 7 healthy male Beagles (body weight, 10.5 to 15 kg; age, 2 to 4 years). All dogs were treated by IV administration of a high dose of MPSS (30 mg/kg). Additional doses of MPSS (15 mg/kg) were administered IV at 2 and 6 hours and then at 6-hour intervals until 48 hours after the initial dose. Blood samples were collected before and 1, 2, 4, 7, and 14 days after completion of the MPSS administrations and used for evaluation of PMN functions. Isolated PMNs were used for assessment of functions, such as adhesion, migration, phagocytosis, and oxidative burst. On days 1, 2, and 4 after completion of MPSS administration, there was a decrease in PMN expression of adhesion markers such as CD11b and CD18. There was a decrease in the phagocytotic ability of PMNs on days 1, 2, and 7 after completion of MPSS administration, with a reduction in the oxidative burst of PMNs detected on day 7. No significant changes were identified for migration. All functional changes returned to their pretreatment values by 14 days after completion of MPSS treatment. Treatment with a high dose of MPSS suppressed PMN functions in dogs. Analysis of these results suggested that treatment with a high dose of MPSS can suppress some of the major functions of PMNs for at least 7 days.

Never Underestimate the Power of a Neutrophil

Polymorphonuclear neutrophil leukocytes (PMNs) have long been viewed as short-lived effector cells capable of phagocytosing pathogens and mediating tissue damage. In this issue of Immunity, Zhang et al. (2009) shed fresh light on the plasticity and immunoregulatory functions of PMNs.

Heifer and quarter characteristics associated with periparturient blood and milk neutrophil apoptosis in healthy heifers and in heifers with subclinical mastitis

Polymorphonuclear neutrophilic leukocytes (PMNL) play an important role in the first line cell-mediated immune defense of the body in general and of the mammary gland against mastitis pathogens in particular. Reduced viability of PMNL close to parturition may explain the high incidence of infectious diseases and the high prevalence of intramammary infections (IMI) in periparturient dairy heifers. Apoptosis of blood PMNL 1 wk before the expected calving date and of blood and milk PMNL at 1 to 4 d in milk was determined using flow cytometry. Information on heifer and gland characteristics was collected before calving and in early lactation. Data were analyzed using multivariable, multilevel regression analysis. Supplementation of a commercial mineral/vitamin mix before calving was associated with less blood (14.4±2.9 vs. 22.4±2.1%) and milk PMNL apoptosis (19.0±1.1 vs. 26.4±0.9%) near calving, presumably related to higher blood selenium concentrations. Both blood and milk PMNL apoptosis showed seasonal variation with the highest proportion of apoptotic cells between January and March (32.0±6.1 and 34.6±2.7%, respectively) and April and June (31.3±5.7 and 37.8±2.3%, respectively). Heifers losing 0.25 points or more of their body condition in the periparturient period had higher proportions of apoptotic blood PMNL in early lactation compared with heifers losing less than 0.25 points (24.0±2.8 vs. 16.6±1.7%). Milk PMNL apoptosis was less pronounced in quarters having teat orifices colonized with non-aureus staphylococci before calving (18.9±1.0 vs. 29.4±1.0%). The variation in blood PMNL apoptosis before and after calving mainly resided at the heifer level (71.4 and 98.4% of the total variation, respectively), whereas the variation in milk PMNL apoptosis mainly resided at the heifer (45.7% of the total variation) and quarter levels (45.5% of the total variation). These data imply that the impaired blood and milk PMNL viability in periparturient heifers can be reduced by optimization of certain heifer management practices such as supplementation of minerals/vitamins, and pasture and feeding strategies.

Self-assembled hemocompatible coating on poly (vinyl chloride) surface

A stable hemocompatible coating was fabricated by consecutive alternating adsorption of iron (III) and two kinds of polysaccharides, heparin (Hep) and dextran sulfate (DS), onto poly (vinyl chloride) (PVC) surfaces via electrostatic interaction. The fluctuation of contact angles with the alternative deposition of iron (III) and polysaccharides verified the progressive buildup of the mulitilayer coating onto the PVC surface. Atomic force microscopy (AFM) analysis revealed that the PVC surfaces were completely masked by iron–polysaccharides multilayer coatings. The activated partial thromboplastin time (APTT) assay showed that both Hep/Fe 3+/Hep and DS/Fe 3+/Hep coated PVC were less thrombogenic than the uncoated one. Chromogenic assay for heparin activity proved definitively that the inhibition of locally produced thrombin was ascribed to the thromboresistance of the surface-bound heparin. Compared with the unmodified PVC surfaces, iron–polysaccharide multilayer coating presented a drastically reduced adhesion in vitro of platelets, polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC). Interestingly, the DS/Fe 3+/Hep coating was found to exhibit higher hydrophilicity and stability, hence lower non-specific protein adsorption in comparison with Hep/Fe 3+/Hep coating due to the incorporation of dextran sulfate into the multilayer coating.