C5a and TNF-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is characterized by the presence of fibrin-rich inflammatory exudates in the intra-alveolar spaces and the extensive migration of neutrophils into alveoli of the lungs. Tissue factor (TF)-dependent procoagulant properties of bronchoalveaolar lavage fluid (BALF) obtained from ARDS patients favor fibrin deposition, and are likely the result of cross-talk between inflammatory mediators and hemostatic mechanisms. However, the regulation of these interactions remains elusive. Prompted by previous findings suggesting that neutrophils, under certain inflammatory conditions, can express functional TF, we investigated the contribution of intra-alveolar neutrophils to the procoagulant properties of BALF from patients with ARDS. Our results confirm that the procoagulant properties of BALF from ARDS patients are the result of TF induction, and further indicate that BALF neutrophils are a main source of TF in intra-alveolar fluid. We also found that BALF neutrophils in these patients express significantly higher levels of TF than peripheral blood neutrophils. These results suggest that the alveolar microenvironment contributes to TF induction in ARDS. Additional experiments indicated that the ability of BALF to induce TF expression in neutrophils from healthy donors can be abolished by inhibiting C5a or TNF-alpha signaling, suggesting a primary role for these inflammatory mediators in the up-regulation of TF in alveolar neutrophils in ARDS. This cross-talk between inflammatory mediators and the induction of TF expression in intra-alveolar neutrophils may be a potential target for novel therapeutic strategies to limit ARDS-associated disturbances of coagulation.     

Leukaemic peripheral blood plasma and bone marrow plasma: comparison of influence on lymphocyte proliferation

Abstract. Peripheral blood plasma from some children with untreated acute lymphoblastic leukaemia (ALL) exerted an inhibitory effect in vitro on phytohaemagglutinininduced lymphocyte transformation of normal peripheral blood lymphocytes. This occurred at concentrations beyond that required for optimal response as judged by reduction of blast cell formation and tritiated thymidine and tritiated uridine incorporation into DNA and RNA, respectively. In contrast, bone marrow plasma from these patients was non-inhibitory or contained significantly less inhibitory activity. Bone marrow plasma from the majority of healthy controls was superior to their peripheral blood plasma in enhancing phytohaemagglutinin-induced mitogenesis. The difference between an individual's bone marrow- and peripheral blood-derived plasma in enhancing proliferation of patient and healthy control cells was significantly greater amongst the patients than the healthy control group; this was attributed mainly to the increased inhibitory activity of ALL peripheral blood plasma compared with normal plasma. Medium conditioned by phytohaemagglutinin-stimulated normal peripheral blood lymphocytes was effective in neutralizing the inhibitory activity of ALL peripheral blood plasma. Taken together, these in vitro results are at least suggestive that in vivo , in healthy subjects, the rapidly proliferating cells in the bone marrow and the 'resting' blood cells in the circulation may be under the influence of a fine balance of different types and/or levels of humoral growth stimulatory and inhibitory factors and that in ALL an unstable balance of these factors exists. The decreased proliferation of circulating blast cells compared with bone marrow blasts in ALL may be attributed, at least in part, to exposure to the different levels of inhibitor(s) in the circulation and bone marrow as demonstrated in vitro by our results.     

嗜中性粒细胞是人抵抗素表达的主要细胞

抵抗素(resistin)是小鼠白色脂肪组织大量表达的富含半胱氨酸的 分泌型蛋白.近年研究发现,人与啮齿类动物的抵抗素组织表达分布存在很 大差异．小鼠抵抗素主要在白色脂肪组织表达,而人抵抗素主要在单核细 胞/巨噬细胞表达,且在骨髓组织中大量表达,但目前骨髓中的细胞定位还 不清楚．本研究的目的是明确成人骨髓及外周血白细胞中抵抗素表达细胞 的类型．免疫荧光法检测骨髓中抵抗素表达细胞,结果显示,抵抗素主要表 达在细胞核呈杆状和分叶核状的成熟粒细胞中,其中杆状核粒细胞表达较 高,分叶核粒细胞表达减弱．Anti-hresistin IgG-Biotin-PE单色荧光流 式细胞术分选外周血白细胞中抵抗素表达细胞后经瑞氏化学染色,结果显 示,抵抗素表达细胞主要为杆状和分叶核状的嗜中性粒细胞,还有少量嗜酸 性粒细胞,且抵抗素蛋白分布在细胞质中. RT-qPCR结果在RNA水平上证明, 人抵抗素在嗜中性粒细胞中大量表达．Anti-hresistin IgG-FITC和anti- HNL IgG-Biotin-PE 双色荧光流式细胞术进一步证明,抵抗素的主要表达细 胞为成熟的嗜中性粒细胞．嗜中性粒细胞在机体免疫防御中起重要作用, 人骨髓及外周血中抵抗素主要在成熟嗜中性粒细胞中表达,这一研究结论 为人抵抗素与炎症反应的关联性及其功能的进一步研究奠定了基础.     

Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation

Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull. We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate. We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine. 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull. We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils. Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process.     

Interleukin 8 (IL-8) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ARDS) and patients at risk for ARDS.

A sensitive and specific radioimmunoassay was used to measure interleukin 8 (IL-8) in bronchoalveolar lavage fluids from control subjects, patients with the adult respiratory distress syndrome (ARDS) and patients undergoing coronary bypass surgery, a risk factor for developing ARDS. Concentrations of IL-8, albumin, total protein and numbers of neutrophils were higher in both patient groups than in controls. Levels of IL-8 were significantly correlated with the influx of neutrophils, plasma protein extravasation and with the PaO2/FiO2 ratio. These data suggest that IL-8 may mediate the recruitment of neutrophils from the vascular compartment into the alveolar space and may therefore be an important determinant in neutrophil-mediated lung injury. Since increased levels of IL-8 were also found in BAL fluid from patients at risk in whom ARDS did not develop, other factors are likely to be involved and IL-8, as well as other markers of inflammation, are of little prognostic use.     

Regulation of hepatocyte growth factor secretion by fibroblasts in patients with acute lung injury

The mechanisms of pulmonary repair in acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are poorly known. Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) are key factors involved in alveolar epithelial repair, present in the bronchoalveolar lavage fluid (BALF) from patients with ALI/ARDS. The role of BALF mediators in their production remains to be determined. We evaluated the overall effect of BALF from 52 patients (27 ventilated patients with ALI/ARDS, 10 ventilated patients without ALI, and 15 nonventilated control patients) on HGF and KGF synthesis by lung fibroblasts. Fibroblasts were cultured in the presence of BALF. HGF and KGF protein secretion was measured using ELISA, and mRNA expression was evaluated using quantitative real-time RT-PCR. Only BALF from ALI/ARDS patients upregulated both HGF and KGF mRNA expression and protein synthesis (+271 and +146% for HGF and KGF, respectively). BALF-induced HGF synthesis from ALI/ARDS patients was higher than that from ventilated patients without ALI (P < 0.05). HGF secretion was correlated with BALF IL-1beta levels (rho = 0.62, P < 0.001) and BALF IL-1beta/IL-1 receptor antagonist ratio (rho = 0.54, P < 0.007) in the ALI/ARDS group. An anti-IL-1beta antibody partially (>50%) inhibited the BALF-induced HGF and PGE(2) secretion, whereas NS-398, a specific cyclooxygenase-2 (COX-2) inhibitor, completely inhibited it. Anti-IL-1beta antibodies as well as NS-398 reversed the COX-2 upregulation induced by BALF. Therefore, IL-1beta is a main BALF mediator involved in HGF secretion, which is mediated through a PGE(2)/COX-2-dependent mechanism. BALF mediators may participate in vivo in the production of HGF and KGF by lung fibroblasts during ALI/ARDS.     

Histamine release and circulating cells after antigen inhalation in allergic dogs

Histamine can be recovered from the blood of ragweed-sensitized dogs after aerosol antigen challenge, although its source is unknown. Neutrophils and eosinophils have been recovered from bronchoalveolar lavage fluid (BALF) obtained under identical conditions. We investigated the time course of changes in histamine levels in plasma and BALF taken from ragweed-sensitized dogs after aerosol challenge. Changes in the numbers of circulating neutrophils, eosinophils, lymphocytes, monocytes, and platelets were also studied. After 3 min, total pulmonary resistance (RL) was maximally increased and systolic blood pressure was maximally decreased. Histamine levels in plasma and BALF were increased and circulating eosinophils and neutrophils were decreased. After 15 min, platelet numbers were reduced. By 90 min, changes in RL, blood pressure, plasma and BALF histamine concentrations, and circulating neutrophils and eosinophils had returned to base-line values, but platelet numbers remained significantly decreased. Sham challenge caused no significant changes in any of these variables. Intravenous administration of histamine in doses large enough to attain plasma levels comparable with those achieved after aerosol antigen challenge resulted in no concomitant rise in BALF histamine levels. We conclude that antigen challenge in sensitized dogs causes increases in BALF and plasma histamine levels and is associated with a reduction in circulating neutrophils, eosinophils, and platelets. It is likely that antigen causes airway mast cells to release mediators that move down a concentration gradient from the airways to the pulmonary circulation.     

Protein C and thrombomodulin in human acute lung injury

Decreased circulating protein C and increased circulating thrombomodulin are markers of the prothrombotic, antifibrinolytic state associated with poor outcomes in sepsis but have not been measured in patients with ALI (acute lung injury)/ARDS (acute respiratory distress syndrome). We measured circulating and intra-alveolar protein C and thrombomodulin in 45 patients with ALI/ARDS from septic and nonseptic causes and correlated the levels with clinical outcomes. Plasma protein C levels were lower in ALI/ARDS compared with normal. Lower levels of protein C were associated with worse clinical outcomes, including death, fewer ventilator-free days, and more nonpulmonary organ failures, even when only patients without sepsis were analyzed. Levels of thrombomodulin in pulmonary edema fluid from ALI/ARDS patients were >10-fold higher than normal plasma and 2-fold higher than ALI/ARDS plasma. Higher edema fluid thrombomodulin levels were associated with worse clinical outcomes. The higher levels in edema fluid compared with plasma suggest local release of soluble thrombomodulin in the lung, possibly from a lung epithelial source. To determine whether lung epithelial cells can release thrombomodulin, A549 cells and primary isolates of human alveolar type II cells were exposed to H2O2 or inflammatory cytokines. Both epithelial cell types released thrombomodulin into the media. In summary, the protein C system is markedly disrupted in patients with ALI/ARDS from both septic and nonseptic causes. The protein C system may be a potential therapeutic target in patients with ALI/ARDS.     

Fc gamma RIIIb allele-sensitive release of alpha-defensins: anti-neutrophil cytoplasmic antibody-induced release of chemotaxins

Antineutrophil cytoplasmic Abs (ANCA) can activate neutrophils in an FcgammaR-dependent manner, but the link between this ANCA-induced effect and mononuclear cell activation with the characteristic granuloma formation of Wegener's granulomatosis is unclear. Human alpha-defensins, small cationic antimicrobial peptides, are found in neutrophils and have chemotactic activity for T cells, dendritic cells, and monocytes. In this study, we quantitated the release of alpha-defensins (human neutrophil peptides 1-3) from human neutrophils after targeted FcgammaR cross-linking (XL). Homotypic XL of FcgammaRIIa, FcgammaRIIIb, or heterotypic XL of both receptors resulted in significant release of alpha-defensins, an effect also induced by both human polyclonal and murine monoclonal cytoplasmic staining ANCA (anti-proteinase 3). This release of alpha-defensins, as well as of other granule constituents (ANCA targets anti-proteinase 3 and myeloperoxidase and elastase), was significantly greater in donors homozygous for the NA1 allele of FcgammaRIIIb than in donors homozygous for NA2. Interestingly, the ANCA-induced release was completely inhibited by the IgG Fc-binding peptide TG19320, which blocks the IgG-Fc region from binding to FcgammaR. Based on their chemotactic properties, alpha-defensins and their release by ANCA may contribute to modulation of the acquired immune response and to granuloma formation. The greater activity of the FcgammaRIIIB-NA1 genotype may also explain the greater severity of disease and its flare-ups in patients with this allele.     

Neutrophil maturation and activation determine anatomic site of clearance from circulation

The long-term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using (111)In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 +/- 1.9 vs. 31.9 +/- 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 +/- 1.1%) and marrow-derived neutrophils to the marrow (65.9 +/- 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence supporting both storage and perhaps disposal functions. Furthermore, models for circulating neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell.     

Action of compounds which predominantly affect the peripheral M-cholinoreactive systems on the bone marrow eosinophils

Experiments on rats have shown that the blockade of M-cholinoreceptors with atropine (50,100 and 150 mg/kg) delays, while the stimulation of the M-cholinoreceptors with aceclidine (5, 10 and 15 mg/kg) intensifies the release of mature eosinophils from the bone marrow to the circulating blood. It is concluded that the peripheral M-cholinoreactive systems participate in the control of the release of the mature bone marrow eosinophils into the blood.     

Cyclophosphamide induced generation of giant hypersegmented granulocytes in rat bone marrow: cell cycle distribution and silver nucleolar staining

Daily injection of cyclophosphamide (20 mg/kg body weight) for 7 days resulted in accumulation of 50% of rat bone marrow granulocytes in G2. Tetraploid neutrophils were hypersegmented (7.25 +/- 0.33) in comparison with diploid ones (3.92 +/- 0.33). After 14 days of cyclophosphamide treatment tetraploid hypersegmented neutrophils could be found in peripheral blood. Diploid neutrophils in these animals were also hypersegmented (4.78 +/- 0.14 versus 3.15 +/- 0.02 in control, p less than 0.001). Nucleolar ribosomal gene activities, evaluated by morphometry of silver nucleolar grains, decreased on bone marrow granulocytes in the course of differentiation in control rats. After cyclophosphamide treatment mature granulocytes contained more silver grains than in controls which may be explained by conservation of silver binding sites of nucleoli from the stages of promyelocytes and myelocytes. These results suggest two mechanisms of hypersegmented neutrophil, generation in cyclophosphamide treated rats: the first, via maturation of myelocytes arrested in G2, and second, a direct one, without tetraploid granulocyte involvement.     

Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow

The mechanisms by which mature myeloid cells are released from the bone marrow into the peripheral blood are not clearly understood. Glycosylation is likely to play an important role, as has been shown in the homing of lymphocytes to lymph nodes and of neutrophils to inflamed endothelia. Cell surface sialylation is an important component of many cellular adhesive interactions, both as ligand-promoting interactions, as occurs in selectin and sialoadhesin-mediated adhesion, and for reducing cell adhesion as in some cancer cells. We have studied the expression of cell surface alpha2,6-linked sialic acid in the maturation of normal bone marrow myeloid cells, the expression of alpha2,6-sialyltransferase mRNA, and the role of sialylation in the adherence of myeloid cells to bone marrow stroma. Our data show that there is a dramatic increase in cell surface alpha2,6-sialylation during the late stage of maturation. This up-regulation is restricted to specific glycoproteins including CD11b and CD18. It is associated with a relative increase in the level of alpha2,6-sialyltransferase mRNA compared with alpha2,3-sialyltransferase mRNA. The changes in mature bone marrow myeloid cells are associated with reduced cell binding to fibronectin and cultured bone marrow stroma. Our data strongly suggest that alpha2,6-sialylation may be important in the interaction between maturing myeloid cells and bone marrow stroma and may govern the release of cells from the bone marrow into the peripheral blood.     

Imbalance in the expression of CXC chemokines correlates with bronchoalveolar lavage fluid angiogenic activity and procollagen levels in acute respiratory distress syndrome

Diffuse alveolar damage is the histopathological hallmark of acute respiratory distress syndrome (ARDS) and is a stereotypic response to a variety of etiologies. Moreover, a significant proportion of ARDS survivors have residual pulmonary fibrosis and compromised pulmonary function. This suggests that the pathogenesis of diffuse alveolar damage that ultimately leads to the chronic fibrosis of ARDS has features of dysregulated repair exemplified by exaggerated intra-alveolar angiogenesis and fibrogenesis (i.e., fibroproliferation and deposition of extracellular matrix), leading to progressive alveolar fibrosis and impaired lung function. We obtained bronchoalveolar lavage fluid (BALF) from patients with ARDS or ventilated control patients and assessed CXC chemokine levels by ELISA. We found an imbalance in the expression of ELR(+) as compared with ELR(-) CXC chemokines from BALF of patients with ARDS as compared with controls. This imbalance correlated with angiogenic activity as assessed by the corneal micropocket assay. Furthermore, these levels correlated with both procollagen I and procollagen III levels in BALF. In contrast, while BALF levels of vascular endothelial growth factor were elevated, vascular endothelial growth factor did not appear to be significantly contributing to the angiogenic activity. These findings suggest that CXC chemokines have an important role in the fibroproliferative phase of ARDS via the regulation of angiogenesis.     

Inhibitors of elastase and cathepsin G in Chédiak-Higashi (beige) neutrophils

Previous studies have established that mature neutrophils from the peritoneal cavity, blood, and bone marrow of beige (Chédiak-Higashi syndrome) mice essentially lack activities of two lysosomal proteinases: elastase and cathepsin G. There are, however, significant levels of each enzyme in early neutrophil precursors in bone marrow. In the present experiments, it was found that the addition of extracts from mature beige neutrophils to extracts of normal neutrophils or to purified human neutrophil elastase and cathepsin G resulted in a significant inhibition of elastase and cathepsin G G activities. 125I-Labeled human neutrophil elastase formed high molecular mass complexes at 64 and 52 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis when added to beige neutrophil extracts. The molecular masses of the inhibitor-125I-elastase complexes suggested that the molecular masses of the inhibitors are approximately 36 and 24 kDa, respectively. These results were confirmed by gel filtration on Superose 12 under nondenaturing conditions. Cathepsin G was inhibited only by the 36-kDa component. The inhibitors formed a covalent complex with the active sites of elastase and cathepsin G. No inhibitory activity was present in mature neutrophil extracts of genetically normal mice or in extracts of bone marrow of beige mice. These results thus represent an unusual example of an enzyme deficiency state caused by the presence of excess inhibitors. Inactivation of neutrophil elastase and cathepsin G in mature circulating and tissue neutrophils may contribute to the increased susceptibility of Chédiak-Higashi patients to infection.     

Impact of neutrophils on antiviral activity of human bronchoalveolar lavage fluid

Surfactant protein D (SP-D) and neutrophils participate in the early innate immune response to influenza A virus (IAV) infection. SP-D increases neutrophil uptake of IAV and modulates neutrophil respiratory burst responses to IAV; however, neutrophil proteases have been shown to degrade SP-D, and human neutrophil peptide defensins bind to SP-D and can cause precipitation of SP-D from bronchoalveolar lavage fluid (BALF). BALF has significant antiviral activity against IAV. We first added neutrophils to BALF during incubation with IAV. Addition of neutrophils to BALF caused significantly greater clearance of IAV from culture supernatants than from BALF alone, and this effect was significantly more pronounced when neutrophils were activated during incubation with the virus. In contrast, if activated neutrophils were incubated with BALF before addition of virus, they reduced antiviral activity of BALF. This effect correlated with depletion of SP-D from BALF. Activation of neutrophils with agonists that induce primary granule release (including release of human neutrophil peptide defensins) caused SP-D depletion, but activation with PMA, which causes only secondary granule release, did not. The ability of activated neutrophils to deplete SP-D from BALF was partially, but not fully, corrected with protease inhibitors but was unaffected by inhibition of neutrophil respiratory burst responses. These results suggest that chronic neutrophilic inflammation (e.g., as in chronic smoking or cystic fibrosis) may reduce SP-D levels and predispose to IAV infection. In contrast, acute inflammation, as occurs in the early phase of IAV infection, may promote neutrophil-mediated viral clearance.     

Homeostatic regulation of blood neutrophil counts

Blood neutrophil counts are determined by the differentiation and proliferation of precursor cells, the release of mature neutrophils from the bone marrow, margination, trafficking and transmigration through the endothelial lining, neutrophil apoptosis, and uptake by phagocytes. This brief review summarizes the regulation of blood neutrophil counts, which is in part controlled by G-CSF, IL-17, and IL-23. Neutrophils are retained in the bone marrow through interaction of CXCL12 with its receptor CXCR4. The relevance of this mechanism is illustrated by rare diseases in which disrupting the desensitization of CXCR4 results in failure to release mature neutrophils from bone marrow. Although blood neutrophil numbers in inbred mouse strains and individual human subjects are tightly controlled, their large variation among outbred populations suggests genetic factors. One example is benign ethnic neutropenia, which is found in some African Americans. Reduced and elevated neutrophil counts, even within the normal range, are associated with excess all-cause mortality.     

Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

Non-Invasive monitoring of diaphragmatic timing by means of surface contact sensors: An experimental study in dogs

Background

The predictive role of many cytokines has not been well defined in Acute Respiratory Distress Syndrome (ARDS).

Methods

We measured prospectively IL-4, IL-6, IL-6 receptor, IL-8, and IL-10, in the serum and bronchoalveolar lavage fluid (BALF) in 59 patients who were admitted to ICU in order to identify predictive factors for the course and outcome of ARDS. The patients were divided into three groups: those fulfilling the criteria for ARDS (n = 20, group A), those at risk for ARDS and developed ARDS within 48 hours (n = 12, group B), and those at risk for ARDS but never developed ARDS (n = 27, group C).

Results

An excellent negative predictive value for ARDS development was found for IL-6 in BALF and serum (100% and 95%, respectively). IL-8 in BALF and IL-8 and IL-10 serum levels were higher in non-survivors in all studied groups, and were associated with a high negative predictive value. A significant correlation was found between IL-8 and APACHE score (r = 0.60, p < 0.0001). Similarly, IL-6 and IL-6r were highly correlated with PaO2/FiO2 (r = -0.27, p < 0.05 and r = -0.55, p < 0.0001, respectively).

Conclusions

BALF and serum levels of the studied cytokines on admission may provide valuable information for ARDS development in patients at risk, and outcome in patients either in ARDS or in at risk for ARDS.