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Background People with human immunodeficiency virus (PWH) have an increased risk of developing chronic obstructive pulmonary disease (COPD). Methods We phenotyped lung macrophages in 4 subgroups—M1 (CD40+CD163−), M2 (CD40−CD163+), double positives (CD40+CD163+), and double negatives and (CD40−CD163−)—and we determined their phagocytic capacity in PWH with and without COPD. Results People with human immunodeficiency virus with COPD have more double-negative macrophages (84.1%) versus PWH without (54.3%) versus controls (23.9%) (P=.004) and reduced phagocytosis (P=.012). Double-negative macrophages had the worst phagocytic capacity (P Conclusions People with human immunodeficiency virus with COPD have an abundance of nonpolarized macrophages, which have poor phagocytic capacity and therefore predispose PWH to increased risk of disease progression.

The classical M1/M2 polarity of macrophages may not be applicable to inflammatory lung diseases including chronic obstructive pulmonary disease (COPD) due to the complex microenvironment in lungs and the plasticity of macrophages. We examined macrophage sub-phenotypes in bronchoalveolar lavage (BAL) fluid in 25 participants with CD40 (a M1 marker) and CD163 (a M2 marker). Of these, we performed RNA-sequencing on each subtype in 10 patients using the Illumina NextSeq 500. Approximately 25% of the macrophages did not harbor classical M1 or M2 surface markers (double negative, DN), and these cells were significantly enriched in COPD patients compared with non-COPD patients (46.7% vs. 14.5%, p

Lung macrophages are the key immune effector cells in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Several studies have shown an increase in their numbers in bronchoalveolar lavage fluid (BAL) of subjects with COPD compared to controls, suggesting a pathogenic role in disease initiation and progression. Although reduced lung macrophage phagocytic ability has been previously shown in COPD, the relationship between lung macrophages&rsquo, phenotypic characteristics and functional properties in COPD is still unclear. (1) Methods: Macrophages harvested from bronchoalveolar lavage (BAL) fluid of subjects with and without COPD (GOLD grades, I&ndash, III) were immuno-phenotyped, and their function and gene expression profiles were assessed using targeted assays. (2) Results: BAL macrophages from 18 COPD and 10 (non-COPD) control subjects were evaluated. The majority of macrophages from COPD subjects were non-polarized (negative for both M1 and M2 markers, 77.9%) in contrast to controls (23.9%, p &lt, 0.001). The percentages of these non-polarized macrophages strongly correlated with the severity of COPD (p = 0.006) and current smoking status (p = 0.008). Non-polarized macrophages demonstrated poor phagocytic function in both the control (p = 0.02) and COPD (p &lt, 0.001) subjects. Non-polarized macrophages demonstrated impaired ability to phagocytose Staphylococcus aureus (p &lt, 0.001). They also demonstrated reduced gene expression for CD163, CD40, CCL13 and C1QA&amp, B, which are involved in pathogen recognition and processing and showed an increased gene expression for CXCR4, RAF1, amphiregulin and MAP3K5, which are all involved in promoting the inflammatory response. (3) Conclusions: COPD is associated with an abundance of non-polarized airway macrophages that is related to the severity of COPD. These non-polarized macrophages are predominantly responsible for the poor phagocytic capacity of lung macrophages in COPD, having reduced capacity for pathogen recognition and processing. This could be a key risk factor for COPD exacerbation and could contribute to disease progression.

Background/Aims: The ideal hemoglobin target in chronic kidney disease remains unknown. Ultimately, individualized targets may depend upon the properties of the patient’s endothelial and vascular milieu, and thus the complex relationships between these factors need to be further explored. Methods: Forty-six patients with a glomerular filtration rate (GFR) 2 or on renal replacement therapy underwent measurement of hemoglobin, endothelial microparticles (EMPs) and aortic pulse wave velocity (PWV) at 0, 3 and 6 months. In addition, a number of inflammatory, cardiac and vascular biomarkers were measured at baseline. Results: No correlation was observed between baseline values of PWV and EMPs, PWV and hemoglobin, or hemoglobin and EMPs in the overall cohort. When stratified by CKD status, a positive correlation was observed between PWV and EMP CD41–/CD144+ in patients with GFR 2 only (r = 0.54, p = 0.01). Asymmetric dimethylarginine correlated with baseline PWV (r = 0.27, p = 0.07), and remained significantly correlated with the 3- and 6-month PWV measurement. Conclusions: In this small heterogeneous cohort of dialysis and non-dialysis patients, we were unable to describe a physiologic link between anemia, endothelial dysfunction and arterial stiffness.

Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5′-bromo-2′-deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN (PMNBrdU) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The PMNBrdUwere assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, PMNBrdUin the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 ± 4.2 h) and maturation pool (65.5 ± 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of PMNBrdUfrom the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior.

The recruitment of monocytes into the alveolar spaces is crucial for clearing infections and resolving the inflammatory response. We have previously reported the effect of acute pneumonia on monocyte transport through the bone marrow, and the present study concerns their clearance from the blood and migration into the lung airspaces. Dividing monocytes were labeled with the thymidine analog, 5'-bromo-2'-deoxyuridine (BrdU). Whole blood containing the labeled monocytes (MO(BrdU)) was transfused from either donor rabbits with pneumonia or from uninfected controls into recipients with pneumonia, where they were detected in blood and tissues using a double immunostaining method. The results show that MO(BrdU) from infected animals rapidly disappeared from the circulation (P0.05), preferentially sequestered in the infected lung tissue within 1 h (22.0 +/- 3.3% versus 6.0 +/- 0.4%, pneumonic region versus peripheral blood, P0.05), and accumulated to a greater degree in pneumonic airspaces than control monocytes 48 h after transfusion (3.9 +/- 0.5% versus 1.1 +/- 0.1%, P0.05). We conclude that immature monocytes released from the marrow by pneumonia sequester rapidly in lung microvessels but their migration in pneumonic airspaces is delayed.

Bacterial endotoxin or lipopolysaccharide (LPS) is an important causative agent of sepsis. This study determines the expression of defensins NP-2 and NP-5 and the function of polymorphonuclear leukocytes (PMN) in rabbits treated with LPS. PMN functional activity was assessed by measuring CD18 expression and H2O2production and by examining the lungs. NP-2 and, to a minor extent, NP-5 of circulating PMN increase during endotoxemia. This early increase is concomitant with neutrophilia and elevated CD18 expression and H2O2production, as well as with enhanced NP-2 immunoreactivity in pulmonary microvessels. A decline in defensins, shortly after the last LPS treatment, is associated with a decrease in the circulating activated PMN and enhanced immunoreactivity in the inflammatory cells, as well as with lung tissue damage. This study shows that LPS-induced changes in the defensins of circulating PMN correlate with the number and activated condition of these cells and suggests that PMN-derived products implement the inflammatory reaction that leads to lung injury and sepsis.

Polymorphonuclear neutrophils (PMN) are vital in host defense against microbial infections. This study provides a flow cytometric method for the quantitative analysis of microbicidal peptides (defensins) in cells of PMN lineage. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing 1-selectin. PMN lineage counts were made on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alkaline phosphatase technique. The results show that marrow PMN express higher levels of NP-2 and NP-5 than blood PMN, p < 0.001 and that these levels are associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence intensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most 1-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surface. Furthermore, defensins are not detected in monocytes, eosinophils, lymphocytes and erythrocytes. The flow cytometric method described here provides a novel means of quantitating host natural defenses, allows the characterization of PMN subpopulations and has clinical applications.

Background Markers of plaque destabilization and disruption may have a role in identifying non-STE- type 1 Myocardial Infarction in patients presenting with troponin elevation. We hypothesized that a plaque disruption index (PDI) derived from multiple biomarkers and measured within 24 hours from the first detectable troponin in patients with acute non-STE- type 1 MI (NSTEMI-A) will confirm the diagnosis and identify these patients with higher specificity when compared to individual markers and coronary angiography. Methods We examined 4 biomarkers of plaque destabilization and disruption: myeloperoxidase (MPO), high-sensitivity interleukin-6, myeloid-related protein 8/14 (MRP8/14) and pregnancy-associated plasma protein-A (PAPP-A) in 83 consecutive patients in 4 groups: stable non-obstructive coronary artery disease (CAD), stable obstructive CAD, NSTEMI-A (enrolled within 24 hours of troponin positivity), and NSTEMI-L (Late presentation NSTEMI, enrolled beyond the 24 hour limit). The PDI was calculated and the patients’ coronary angiograms were reviewed for evidence of plaque disruption. The diagnostic performance of the PDI and angiography were compared. Results Compared to other biomarkers, MPO had the highest specificity (83%) for NSTEMI-A diagnosis (P0.05 for all comparisons). The PDI had higher specificity and accuracy for NSTEMI-A diagnosis compared to coronary angiography (P

Aim: To investigate the influence of maternal corticosteroid administration on neutrophil apoptosis in early onset preeclampsia. Methods: We investigated five groups: early onset preeclampsia (EOPET

The elderly are susceptible to infections and show a decline in neutrophil (PMN) functions that are regulated by cytosolic calcium [Ca2+]i. This study measures [Ca2+]i in suspended and adherent PMN of young and elderly individuals by using flow cytometry, confocal microscopy, the bacterial peptide fMLP, and the fluorescent Ca2+ indicator fluor-3/acettoxymethyl ester. PMN from both age groups show a steep and transient fMLP-induced Ca2+ increase. This increase is independent of external divalent cations and is desensitized by a subsequent exposure to the same agonist. Adherent PMN of the elderly express elevated [Ca2+]i before (basal) and after fMLP activation but show reduced ability to mobilize Ca2+ into and from the cytosol. PMN of the elderly take longer (13.7±3 s) to attain the maximal response compared to those of young adults (5.7±0.8 s). PMN from both age groups show heterogeneity in the time and magnitude of this response. However, PMN of the elderly show a decrease in the proportion of cells with prompt and effective reaction and an increase in the representation of a cell subpopulation manifesting delayed response. We conclude that age-related delayed and reduced PMN response to a bacterial peptide could hamper functional activities that are essential in host protection against infections.

Accelerated granulocytopoiesis and polymorphonuclear leukocyte (PMN) activation via the complement system are important events in the inflammatory response. This study tests the hypothesis that PMN of the peripheral blood and the bone marrow behave differently when stimulated with zymosan-activated plasma (ZAP). PMN were treated with ZAP and processed to determine the content and distribution of F-actin, the expression of CD18 integrins, and the cell area/shape with the use of a combination of flow cytometry, fluorescence, and light microscopy. The results show that untreated bone marrow PMN display similar F-actin content and cell area but express less CD18 than peripheral blood PMN. ZAP-activated peripheral blood PMN show a marked increase in the F-actin content, CD18 expression, cell area, and length. Highly elongated PMN develop a polar shape in which microfilaments are redistributed in cell protrusions and CD18 is located in the uropod and the lamellipodium. In comparision, activated bone marrow PMN show a lower and more transient increase in the F-actin content, express less CD18, show a reduced increase in the cell area, and display reduced polarity and microfilament redistribution. We conclude that bone marrow PMN show lower complement-activation response than peripheral blood PMN and suggest that the lower expression of surface membrane integrins and related cytoskeletal filaments may account for their reduced ability to develop the polar shape required for diapedesis and migration.