Assessment of Neutrophil Apoptosis.

The process of neutrophil apoptosis has an important role in the resolution of acute inflammation. Apoptotic cell death is characterized by a coordinated sequence of cellular alterations that serve to uncouple neutrophil effector functions whilst maintaining plasma membrane integrity. In this way the release on neutrophil intracellular contents, including proteases, glycosidases, and reactive oxygen species, is limited during apoptosis. In addition, plasma membrane alterations associated with neutrophil apoptosis provide molecular cues that enable recognition by phagocytic cells, including macrophages. The recognition and uptake of apoptotic neutrophils by macrophages dampens proinflammatory responses to pathogen- or damage-associated molecular patterns and triggers release of proresolution mediators, that further promote resolution of inflammation. The key cellular and molecular events that act to control neutrophil apoptosis and subsequent macrophage phagocytosis have been characterized by in vitro studies, unveiling potential therapeutic targets for the manipulation of these regulatory pathways. In this chapter, we outline some of the key assays that are used to assess neutrophil apoptosis in vitro, together with methods to assess activation of the apoptotic machinery and phagocytic clearance of apoptotic neutrophils.

New flow cytometry-based method for the assessment of the antibacterial effect of immune cells and subcellular particles.

Techniques currently used for assessment of bacterial count or growth are time-consuming, offer low throughput, or they are complicated or expensive. The aim of the present work was to elaborate a new method that is able to detect the antibacterial effect of cells, subcellular particles, and soluble compounds in a fast, cost, and labor effective way. Our proposed technique is based on flow cytometry (FC) optimized for detection of small particles and on fluorescently labeled bacteria. It allows direct determination of the bacterial count in 3 hours. The effect of various human phagocytes and extracellular vesicles on gram-positive and gram-negative bacteria is investigated in parallel with the new, FC-based method, with colony counting and with our previous, OD-based method. Comparing the killing effect of wild type and NADPH oxidase-deficient murine neutrophils presents an example of detection of a clinically important deficiency. Strong correlation was obtained between the results of the different techniques, but the reproducibility of the FC-based test was superior to the OD-based test. The major advantages of the new technique are: rapidity, low cost, high throughput, and simplicity.

Assessment of Post-Vaccination Phagocytic Activation Using Candida albicans Killing Assays.

Candida albicans is an important opportunistic fungal pathogen. It is now the fourth leading cause of nosocomial bloodstream infections and a great threat to the immuncompromised patients attributed to the disseminated candidiasis with the mortality up to 40%. Phagocytic cells are the first line of defense against Candida infections. Antibodies induced by vaccination can effectively enhance the capacities of phagocytosis and killing of neutrophils and macrophages. In this chapter, flow cytometric analysis (FACS) and killing assay by plate culture methods are introduced to evaluate the phagocytosis and killing of strains of Candida albicans opsonized with immune serum obtained from mice vaccinated with yeast and recombinant enolase.

[EXPERIMENTAL ASSESSMENT OF THE IMMUNOTROPIC EFFECTS OF ARABINOGALACTAN AND PECTINACEOUS POLYSACCHARIDES ISOLATED FROM FERULA KUCHISTANICA.]

It is established that arabinogalactan and pectinaceous polysaccharides isolated from Ferula kuchistanica are capable of stimulating a primary immune response in mice by increasing the number of antibody-producing cells in the spleen in response to immunization with sheep red blood cells in both intact animals (on average by 51.0%; p < 0.005) and those with secondary immunodeficiency caused by irradiation (on average by 164.4%; p < 0.005). The treatment with compounds studied also significantly increased the functional condition of cells of the mononuclear phagocyte system (on average by 27.0%; p < 0.005).

Assessment of atherosclerosis in chronic granulomatous disease.

BACKGROUND: Patients with chronic granulomatous disease (CGD) experience immunodeficiency because of defects in the phagocyte NADPH oxidase and the concomitant reduction in reactive oxygen intermediates. This may result in a reduction in atherosclerotic injury. METHODS AND RESULTS: We prospectively assessed the prevalence of cardiovascular risk factors, biomarkers of inflammation and neutrophil activation, and the presence of magnetic resonance imaging and computed tomography quantified subclinical atherosclerosis in the carotid and coronary arteries of 41 patients with CGD and 25 healthy controls in the same age range. Univariable and multivariable associations among risk factors, inflammatory markers, and atherosclerosis burden were assessed. Patients with CGD had significant elevations in traditional risk factors and inflammatory markers compared with control subjects, including hypertension, high-sensitivity C-reactive protein, oxidized low-density lipoprotein, and low high-density lipoprotein. Despite this, patients with CGD had a 22% lower internal carotid artery wall volume compared with control subjects (361.3±76.4 mm(3) versus 463.5±104.7 mm(3); P<0.001). This difference was comparable in p47(phox)- and gp91(phox)-deficient subtypes of CGD and independent of risk factors in multivariate regression analysis. In contrast, the prevalence of coronary arterial calcification was similar between patients with CGD and control subjects (14.6%, CGD; 6.3%, controls; P=0.39). CONCLUSIONS: The observation by magnetic resonance imaging and computerized tomography of reduced carotid but not coronary artery atherosclerosis in patients with CGD despite the high prevalence of traditional risk factors raises questions about the role of NADPH oxidase in the pathogenesis of clinically significant atherosclerosis. Additional high-resolution studies in multiple vascular beds are required to address the therapeutic potential of NADPH oxidase inhibition in cardiovascular diseases. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01063309.

Host defence against disseminated Candida albicans infection and implications for antifungal immunotherapy.

The different manifestations of Candida albicans infection are dictated by an underlying defect in the immune response of the host. Protective immunity to disseminated candidiasis, the manifestation of C. albicans infection discussed in this review, has traditionally been ascribed to innate immunity with emphasis on the role of granulocytes. Lately, however, immunological studies have learned that host defence against disseminated candidiasis is based on a complex interplay between innate and cell-mediated immunity. Despite the availability of new antifungal agents, mortality associated with disseminated C. albicans infection remains high. Immunotherapy that augments host defence is an important strategic option in the battle against disseminated candidiasis. Here, the authors review the chronological events in the pathogenesis of disseminated candidiasis that aid in predicting the impact of existing immunotherapy and the development of future immunomodulating strategies.

Assessment of phagocyte functions by flow cytometry.

Phagocytes neutrophils, monocytes, and macrophages are crucial in the defense against infection. Their functions include phagocytosis, intra- and extracellular digestion of targets, oxidative burst, and chemotaxis. This extensive, detailed unit outlines a four-part procedure for in-depth investigation of these four functions that reflect the attacking and processing of pathogenic microorganisms. Written by the original instigator of flow-based assays in this area, this unit defines an area that is as old as flow itself. Numerous support protocols provide preparative procedures for bacterial targets, opsonins, and antigen-coated beads.

[The phagocytic test in the assessment of antimicrobial protection in inflammatory urologic diseases]. / Fagotsitarnyi test v otsenke antimikrobnoi zashchity pri vospalitel'nykh urologicheskikh zabolevaniiakh.

As shown in in vitro original modelling of red cell action on phagocytic process in the whole blood of patients with urological inflammation, these patients have increased count of neutrophils involved in phagocytosis, but low specific absorption of these neutrophils due to red cells. In chronic renal insufficiency characterized by a low phagocytic activity of neutrophils only stimulating potential of red cells can be noted.

Assessment of phagocytic function using chemiluminescence.

Chemiluminescence is a simple and reliable method of assessing phagocytic function. The bactericidal properties of phagocytes are dependent on the production of powerful oxidising species by the respiratory burst. These reactive oxygen radicals react with biological substrates to form excited compounds which then relax to their ground state by photon emission. This energy release is in the form of light which can be amplified by chemiluminescent probes and measured in a luminometer. Activation of cells is achieved using various agents that stimulate the respiratory burst. There is a close correlation between chemiluminescence and other methods of assessing phagocytic function, including bactericidal ability. The technique can be used to assess the function of polymorphonuclear leukocytes, monocytes, and tissue macrophages in response to disease, drugs, and toxins. This article describes the theory of cellular chemiluminescence, and the use of chemiluminescent probes and various cellular stimuli. Practical aspects of cell isolation and factors affecting chemiluminescence are considered. Finally, the clinical applications of chemiluminescence are discussed.

Immunological function and nutritional assessment.

Theoretically, a large number of tests of immunologic functions could be used for nutritional assessment. However, many of these immunologic tests require specialized laboratory skills and take a long time to perform. These tests provide little additional information to the clinician concerning the nutritional status of the patient, compared to the data that can be obtained from a few simple, selected immunologic measurements. Only a few immunologic tests are sufficiently simple, reproducible, and reliable indicators of nutritional status to be of practical value for routine nutritional assessment. These are the total lymphocyte count and skin tests. At present, all of the other immunologic measurements that have been reviewed should be considered as research tools for nutritional assessment. Immunological tests can be affected by many clinical variables unrelated to nutrition, such as specific pathologic conditions, immunodepressive therapies, accidental or surgical trauma, and infection, and this fact should be considered when using immunological tests to assess nutritional status. Malnutrition suppresses the acute-phase response of plasma proteins. The measurement of the acute-phase response of selected acute-phase proteins can be a functional measurement of nutritional status.

A critical assessment of fibronectin's opsonic role for bacteria and microaggregates.

Within recent years, the physiological roles of fibronectin (Fn) have begun to be elucidated. This review examines Fn's opsonic capabilities with respect to bacteria and microparticulates. Fn's ability to promote the phagocytosis of these targets by monocytes, macrophages, and neutrophils is discussed in detail, as are the possible mechanisms through which Fn mediates these interactions. The controversies concerning the physiological importance of Fn for host homeostasis and its use as an indicator of reticuloendothelial system function are also addressed in this review.

Qualitative and quantitative assessment of C3-receptor reactivities on lymphoid and phagocytic cells.

Highly purified human C3, free of C5 and beta 1H, was used to prepare EAC14oxy23b, EAC14oxy23b' (C3b cells treated with purified C3bINA and beta 1H) and EAC14oxy23d (C3b' cells treated with trypsin). These intermediates were used to assess by rosette formation C3-receptor activity on various cells. The number of cell-bound C3 per C3b, C3b', and C3d cell was quantified by applying 14C-formaldehyde-labeled C3. Human PBL reacted to about the same degree with C3b, C3b', and C3d cells, whereas monocyte-free PBL enriched for B cells interacted preferentially with the C3b' and the C3d cells; human tonsil lymphocytes behaved similarly. The reaction of Raji cells was clearly assessable with C3b cells and was accelerated with C3b' and C3d cells. Daudi cells reacted with C3b' and C3d cells only, in comparison to Raji cells with a much lower activity. Human granulocytes reacted equally well with C3b and C3b' cells, but towards C3d cells they were almost unreactive. Human monocytes formed rosettes with C3b cells, and at a lower level, rosettes with C3b' cells. C3d cells were unreactive. Similar reaction patterns were obtained with guinea pig leukocytes, whereas mouse leukocytes were totally different, since peritoneal macrophages only formed rosettes with human C3b' cells.

A new assay for the assessment of staphylococcal killing by human leucocytes.

A new method is described for investigating the killing of Staphylococcus aureus by human phagocytic cells. The radioassay is based on the principle that only viable bacteria synthesize DNA and incorporate [3H]thymidine. Phagocytes are incubated with bacteria and then disrupted by a single freeze-thaw cycle. The uptake of tritiated thymidine by the remaining organisms is a measure of the killing ability of the phagocytes. The technique is simple, sensitive, rapid and requires small volumes of blood. It can be semiautomated and uses equipment readily available in an immunology laboratory and is therefore suitable for routine investigations of leucocyte function. It is likely that the technique could form the basis for measuring kill by phagocytes of any rapidly dividing organism. A time course and a normal range have been evaluated for the bactericidal capacity of 26 normal individuals.

An assessment of intratumor phagocytic and surface marker-bearing cells in a series of autochthonous and early passaged chemically induced murine sarcomas.

Single cell suspensions from five different 3-methylcholanthrene-induced tumors in CBA mice were examined in the autochthonous host and sequentially for 5-11 passages. They were also examined for Fc receptor-bearing, phagocytic, theta antigen-positive, and surface immunoglobulin-bearing cells. The preparations contained a high proportion of phagocytic and marker-bearing cells both in the original host and during early passage. This proportion was consistent for any particular tumor and passage. Between different tumors, however, the proportions were sufficiently different to allow the tumor to be identified on this basis; this suggested that various chemically induced tumors may be unique in their tumor-host relationship as measured by the type of cells which infiltrate them. With on-going early passage of the tumors, the proportion of marker-bearing cells decreased to a constant level in most instances, mainly because of a reduction in the percentage of phagocytic cells. The tumor with the least macrophages (MBQA, less than 5%) consistently appeared more rapidly and killed the host more rapidly than did the tumor with the most macrophages (MBQD, 15-30%), but was not significantly different in its growth rate. The theta antigen- and Fc receptor-positive cells within these tumors were derived from the animal receiving the tumor inoculum, and thus represented host cell infiltration of the tumor. The results were discussed with reference to fundamental concepts of the immunology of chemically induced tumors and the importance of host cell infiltration within these tumors.