Use of whole blood in the measurement of neutrophil migration.

A whole-blood method was used for measuring neutrophil migration in micropore membrane. The method is reproducible (coefficient of variation (CV) 10.3% and 9.8% in two normal individuals tested repeatedly) and can be performed on an 0.25 ml lithium heparin blood sample. Migration is independent of leucocyte and erythrocyte counts and is comparable with that obtained for separated cells. As in separated cell techniques, cytochalasin and casein respectively inhibit and stimulate neutrophil migration. The technique is of value when testing large numbers of samples and when only small volumes of blood can be obtained, for example, in neonates.     

Enhancement of leukocyte response to lipopolysaccharide by secretory group IIA phospholipase A2.

Secretory nonpancreatic group IIA phospholipase A2 (sPLA2), a lipolytic enzyme found in plasma, is thought to play an important role in inflammation. In patients with sepsis, a strong positive correlation is observed between the plasma level of sPLA2 and poor clinical outcome in sepsis. We have thus asked whether sPLA2 could play a role in enabling responses of cells to bacterial lipopolysaccharide (LPS), a key contributor to sepsis. In the presence of sPLA2, cellular responses to LPS were significantly increased. This was demonstrated in assays of LPS-stimulated interleukin-6 (IL-6) production in whole blood and binding of freshly isolated human polymorphonuclear neutrophils (PMN) to fibrinogen-coated surfaces. We further found that sPLA2 enhanced binding of labeled LPS to PMN, and that the sPLA2-mediated cell responses to LPS were all blocked by monoclonal antibodies directed against membrane CD14. Two properties ofsPLA2 may contribute to its activity to mediate responses to LPS. sPLA2 appears to bind LPS because pre-exposure of sPLA2 to LPS led to a dose-dependent increase in its ability to hydrolyze phospholid substrate, and incubation of sPLA2 with BODIPY-LPS micelles resulted in enhanced fluorescence, presumably from the disaggregation of the LPS aggregates. Additional studies demonstrated that the esterolytic function of sPLA2 is also needed both for the disaggregation of LPS and CD14-dependent cell stimulation. The precise mechanisms by which LPS-binding and esterolytic activity contribute to sPLA2 activity are not clear but our data strongly suggest that these activities result in interaction of LPS with CD14 and subsequent cell activation.     

Cytomegalovirus infectivity in whole blood following leukocyte reduction by filtration.

Cytomegalovirus (CMV) may be transmitted by transfusion of whole blood and cellular components processed according to standard processing procedures. A need exists to develop new procedures to remove CMV and other leukocyte-borne viruses from donor blood. Ten patients (AIDS/bone marrow transplants) who were CMV antigenemic (virus subsequently confirmed by isolation), donated 50 mL of venous blood within 24 to 72 hours of the initial antigen detection. Twenty-five-milliliter aliquots of each specimen were passed through Purecell Neo Neonatal Leukocyte Reduction Filters (Pall, East Hills, NY). The remaining 25-mL nonfiltered aliquots, as well as the blood filtrates, were subjected to infectivity endpoint determinations. The Purecell Neo filter effected a 3 to 4 log10 leukocyte reduction. CMV input titers ranged from less than 10 to 7.3 x 10(1) median tissue culture infectious dose (TCID50) per milliliter. CMV was not isolated from any postfiltration effluent (i.e., leukocytes, erythrocytes, or plasma). CMV DNA was not detected by nested polymerase chain reaction in 8 of 10 postfiltrate blood specimens. The Purecell Neo filter was efficacious in eliminating or significantly reducing viral (CMV) load in venous blood.     

Polymorphonuclear leukocyte response to stimulation in vitro during pregnancy

Oxidative metabolism of polymorphonuclear leukocytes (PMNLs) isolated from pregnant women in the third trimester and from controls were studied using zymosan-induced chemiluminescence (CL) and f-Met-Leu-Phe-stimulated superoxide (O ₂ ^– ) generation. CL was significantly increased during pregnancy, but a decrease was noted in cytochromec reduction. Total cellular levels of -glucuronidase and lysozyme were diminished in PMNLs from pregnant subjects, with unaltered concentrations of cytosol lactate dehydrogenase. The capacity of PMNLs from pregnant women to degranulate did not differ from controls. It is suggested that during pregnancy, in vivo stimulation of PMNLs may occur to account for these changes.     

Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways

Objective:The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia.Materials and methods:The presence of these molecules and the number of proliferating (Ki-67-positive) epithelial cells was analyzed by immunohistochemistry in bronchial epithelium from subjects with (n = 15) or without (n = 14) chronic obstructive pulmonary disease (COPD).Results:Our data demonstrate higher numbers of defensin-positive (p = 0.0001), elastase-positive (p = 0.0001) and Ki-67-positive (p = 0.0001) cells in areas with squamous cell metaplasia as compared to areas with intact or damaged epithelium, while the reverse was observed for SLPI expression (p = 0.002). No differences were observed between subjects with or without COPD, nor between current smokers and those that had stopped smoking.Conclusions:These data are in line with a role of defensins in the hyperproliferative phenotype of squamous metaplastic lesions in the airways. This role does not seem to be restricted to patients with COPD.Received 25 September 2003; returned for revision 3 November 2003, accepted by W. van den Berg 22 December 2003     

Study of the lymphocytein vitro response to rubella antigen and phytohemagglutinin by a whole blood method

Summary A whole blood culture method was used to study lymphocytein vitro responses to rubella antigen and to phytohcmagglutinin (PHA) in rubella infection. The acute phase of infection in four cases was characterized by high spontaneous incorporation of¹⁴C-thymidine in the cultures, unresponsiveness of lymphocytes to rubella antigen, and absence of response, or relatively low response, to PHA. Cells showing vigorousin vitro response to rubella antigen appeared at about two weeks after the onset of rash. Lymphocyte PHA response returned to normal by day 31. Three rubella vaccinees exhibited a similar response. The use of whole blood lymphocyte cultures stimulated with multiple doses of mitogen and with antigen appears to be a promising technique for studies of general and specific cell-mediated immunity in viral infections.With 4 Figures     

Blastogenic response of whole blood cells of turkeys to a T-cell mitogen.

Cellular immune mechanisms in the turkey are not well understood because adequately standardized, reproducible in vitro assays to measure cellular immunity are not available. Our purpose was to optimize conditions for a whole blood mitogenic assay that would facilitate quantitative assessment of the ability of circulating T cells of turkeys to respond to Con A. Heparinized peripheral blood from normal turkeys was examined. Data indicated that diluting the blood 1:20 or 1:40 and incubating the test cultures at 39 degrees C or 41 degrees C gave the best mitogenic stimulation. Presence of 7.5% turkey serum but not chicken or fetal bovine serum in the culture medium substantially enhanced the blastogenic response. Ontogeny of the whole blood mitogenic response was examined by repeat observations on a group of turkeys at various age levels starting at 1 week of age. Whole blood cells from 1-week-old turkeys responded poorly to Con A, although by 2 weeks of age, the response was well developed. Tests at subsequent ages revealed variable levels of activity. There was a considerable individual variation in the level of blastogenic response of turkeys within the same age group.     

Indirect capture augments leukocyte accumulation on P-selectin in flowing whole blood

Leukocytes are captured directly by E- and P-selectin on activated endothelium and by indirect means, which includes attached leukocytes capturing free-flowing leukocytes. However, controversy exists as to whether the latter mechanism occurs in the presence of red blood cells. We analyzed leukocyte capture mechanisms on P-selectin under circulatory hydrodynamics using whole blood. The selective disruption of leukocyte-leukocyte interactions with an L-selectin monoclonal antibody reduced leukocyte accumulation by >50% under various stringencies (substrate concentrations and shear stresses). In addition, a direct analysis of leukocyte capture events revealed that 69% were indirect. Our data indicate that in the presence of red blood cells, P-selectin-attached leukocytes, individually and as a monolayer, augment leukocyte accumulation by indirect capture. This mechanism may contribute to increasing the density of leukocytes on discrete areas of activated endothelial cells at sites of inflammation. These findings are significant since L-selectin accounts for the majority of the leukocyte rolling flux in small venules at diverse inflammatory settings. Yet, the primary mechanism by which L-selectin mediates leukocyte accumulation remains unresolved.     

Fibrinogen-neutrophil interactions in response to fMLP and Porphyromonas gingivalis fimbrial peptides

Porphyromonas gingivalis (P.g) is the primary bacterial agent in many forms of chronic periodontitis. Since polymorphonuclear leukocytes (PMNs) are first-line responders to P.g.- induced inflammation, and fibrinogen is important for in vivo PMN in this disease, we have studied the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP) (an inflammatory stimulus), P.g. fimbriae and fimbrial peptides (based on FimA, the main structural protein of P.g. fimbriae) on PMN-fibrinogen interactions. Freshly isolated human PMNs were allowed to react with FITC-Fibrinogen and various fimbrial peptides (denoted as FimA followed by amino acid number within whole FimA protein), and FITC-Fibrinogen binding was measured using flow cytometry. Freshly isolated neutrophils were also challenged with Fibrinogen and/or fimbrial peptides to measure IL-8 secretion using ELISA. Our studies show that fibrinogen binding to PMNs is enhanced (p < 0.01) in response to fMLP as well as fimbrial peptides (FimA 61-80) containing the motif LTTE (p < 0.01) in a dose dependent manner but not in response to peptides without that motif. We also observed that fMLP and FimA 61-80 have an additive effect on fibrinogen binding to PMNs (p < 0.05), and fMLP and FimA 171-185 significantly inhibit fMLP-induced fibrinogen binding (p < 0.01). To determine of the role of inflammatory cytokines, we examined IL-8 release from PMNs in response to combinations of P. gingivalis fimbriae, fMLP and fibrinogen. In all cases, IL-8 release increased in a dose-dependent manner (p < 0.05). fMLP-fibrinogen effect on IL-8 release from PMNs was synergistic while fimbriae-fibrinogen effect was additive. In summary, PMN priming by fimbrial peptides facilitates fibrinogen-PMN interaction and may increase inflammation.     

The mixed lymphocyte response in whole blood: technical aspects.

Experiments were conducted to standardize the response of lymphocytes in whole blood in mixed culture with allogeneic lymphocytes. The following conditions were found suitable: (1) A culture period of 6-8 days. (2) The ratio of stimulator to responder lymphocytes should be 4-8, but may vary from one batch of stimulator cells to another. (3) Tests may be performed in culture tubes with 50-100 microliter blood in a total volume of 2 ml (macrotechnique) or in microplates with 10 microliter of blood in a volume of 0.2 ml (microtechnique). (4) Serum supplement is not required. (5) Results should be expressed as counts/min (cpm) per a given number of responder lymphocytes. Stimulation indices are less reliable.     

Chemotactic factor induced neutrophil shape changes in whole blood. A comparison of adults and neonates

To define further the nature of the decreased responsiveness of neonatal neutrophils to chemotactic factor stimulation, neutrophil shape change induced by various concentrations of N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) was studied using a whole blood assay. Samples from 48 full term neonates and paired healthy adult controls were examined. The neutrophil response to the chemotactic peptide was assessed by the morphologic transformation from a spherical to a bipolar shape in monolayer blood smears made from fresh whole blood samples. Neonatal neutrophils were found to have increased responsiveness relative to adult controls at low concentrations of fMLP (10(-11) to 10(-9) M), resulting in a significantly lower calculated chemotactic peptide concentration necessary for a 50 percent maximal response (ED50) in neonatal cells (1.01 X 10(-9) M compared to 2.25 X 10(-9) M). The maximal response at higher concentrations of fMLP (5.0 X 10(-9) to 10(-6) M) showed no differences between neonatal and adult cells, thereby supporting the concept that the early cellular events of the chemotactic factor activation in neonatal neutrophils are functionally intact.     

Microfluidic aqueous two phase system for leukocyte concentration from whole blood

Leukocytes from a whole blood sample were concentrated using a microfluidic aqueous two phase system (μATPS). Whole blood was simultaneously exposed to polyethylene glycol (PEG) and dextran (Dex) phase streams and cells were partitioned based on their differential affinity for the streams. The laminar flow characteristic of microfluidic devices was used to create zero, one, and two stable interfaces between the polymer streams. Three different patterns of three polymer streams each were evaluated for their effectiveness in concentrating leukocytes: immiscible PEG-PEG-Dex, immiscible Dex-PEG-Dex, and miscible PEG-PBS-Dex. The most effective configuration was the Dex-PEG-Dex stream pattern which on average increased the ratio of leukocytes to erythrocytes by a factor of 9.13 over unconcentrated blood.     

体外免疫原快速激活全血细胞固有免疫反应系统模式的应用

<正>血液循环的血液固有免疫系统由血浆补体固有免疫子系统、红细胞固有免疫子系统、血小板固有免疫子系统和白细胞固有免疫子系统构成[1]。若采用系统生物学的复杂理论     

A micromethod for stimulation of chicken lymphocytes in vitro using whole blood.

A simple, rapid and reproducible micromethod for determination of in vitro mitogenic responses of chicken peripheral blood lymphocytes is described. The test utilizes 5 mul volumes of heparinized whole blood, 125I-labelled 5-iodo-2'-deoxyuridine with 5-fluoro-2'-deoxyuridine in place of [3H]thymidine and a multiple cell-culture harvester. The method permits a close follow-up of the mitogenic responses in the chicken.     

A whole blood assay to assess peripheral blood dendritic cell function in response to Toll-like receptor stimulation

We report a practical technique to assess peripheral blood dendritic cell (DC) maturation and function in whole blood (WB), which requires minimal blood volumes and minimizes ex vivo manipulations of clinical specimens. We determined optimal conditions for flow cytometric analysis of markers of DC maturation, including CCR7, CD25, CD80 and CD83, and of the intracellular cytokines, tumor necrosis factor- (TNF-) and interferon- (IFN-), in both myeloid (mDC) and plasmacytoid (pDC) lineages. We demonstrate concentration-dependent production of these cytokines by DC following short-term stimulation with ligands to Toll-like receptors (TLRs) 2/1, 3, 4, 7, 8 and 9. Kinetic studies revealed maximal TNF- and IFN- protein expression at 2 to 3 h after stimulation with certain TLR ligands. Finally, utilizing cells from a cohort of eight healthy donors, we compared DC responses to TLR activation in WB, freshly isolated peripheral blood mononuclear cells (PBMC) and cryopreserved PBMC. We found that TNF- responses were essentially preserved, but IFN- responses were profoundly diminished or entirely abrogated following cryopreservation. In conclusion, we propose that WB analysis of peripheral DC function is a rapid, reliable and simple method to evaluate TLR function in clinical specimens, which obviates artifact-prone cell purification. The major impact of cryopreservation on some DC responses further strengthens the case for a rapid method that uses fresh blood.     

A micromethod for in vitro assay of lymphocyte proliferation in mouse whole blood.

A microplate culture system to assay the in vitro proliferative responses of mouse peripheral blood lymphocytes to phytohaemagglutinin is described. The method utilises whole blood, a gamma-emitting isotopic label and semi-automated harvesting. Data are presented to show the effects of several variables on the culture system. The technique is suitable for serial study of the in vitro phytohaemagglutinin response in individual animals.