The phagocytosis capacity assay: a new radiometric phagocytosis test for clinical use

A radiometric technique is described for measuring phagocytosis which does not require separation of extra- and intracellular microorganisms. Following phagocytosis of optimally opsonized yeast cells (Saccharomyces cerevisiae) by mononuclear cells (MNC) or granulocytes, yeast cells remaining extracellular are labeled with [⁷⁵Se]L-selenomethionine. To measure the phagocytic capacity of the cell population investigated, 1–4×10⁶ leukocytes are incubated with 5, 10, 20, 30 and 40×10⁶ yeast cells/ml for testing. Depending on the actual number of phagocytes in the cell populations investigated, different optimal yeast cell concentrations may be determined. Provided measurements are carried out at these optima even moderate effects of therapy can be detected. In a preliminary study of 22 patients with Hodgkin's disease it was shown, that the phagocytic activity of the MNC fraction was higher, but the activity of granulocytes lower, than that of the healthy controls. After combined chemotherapy the above mentioned values returned to normal. This simple, exact method is recommended for clinical use.     

The use of luminous bacteria for determination of phagocytosis

A system has been developed for the isolation of large numbers of unfractionated mononuclear cells from single, well characterized normal individuals and for the separation by elutriation of these cells into populations of greater than 90% pure monocytes and greater than 99% pure lymphocytes. The total number of monocytes obtained from a single donor averaged about 550 million. After cryopreservation and thawing of these cells, the viability remained greater than 90%, 80% of original cells were recovered, and the ability to ingest antibody-coated targets was comparable to that of fresh monocytes. The cells remained sterile without the use of antibiotics and were suitable for long-term culture. The monocytes that were isolated and cryopreserved by these procedures functioned reproducibly as inhibitors of tumor cell growth and in an assay of responsiveness to monocyte migration inhibitory factor (MIF).     

The use of pH-triggered leaky heterogeneities on rigid lipid bilayers to improve intracellular trafficking and therapeutic potential of targeted liposomal immunochemotherapy

During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n = 18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.     

Role of alpha-hemolysin for the in vitro phagocytosis and intracellular killing of Escherichia coli

The role of alpha-hemolysin for the elimination of Escherichia coli by phagocytes in vitro was investigated using sets of isogenic strains which included wild-type alpha-hemolytic strains, derived strains with a reduced production of alpha-hemolysin and derived nonhemolytic strains. Phagocytosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition techniques. alpha-hemolytic strains were phagocytosed and killed to a lesser extent than isogenic strains with a reduced production of alpha-hemolysin and isogenic nonhemolytic strains. The results obtained with granulocytes were similar to those obtained with monocytes although the elimination of bacteria by monocytes was less than that by granulocytes. These results strongly suggest that production of alpha-hemolysin is a means by which E. coli counteracts the activity of phagocytes by injuring these cells with the toxin.     

Removal of surface adherent Staphylococcus aureus in the determination of phagocytosis and intracellular killing by the use of lysostaphin

Use of lysostaphin to remove non-phagocytized, granulocyte-adherent bacteria allows more precise calculation of the percentage of the ingested as well as killed bacteria in the process of phagocytosis.     

The role of iron in infections with intracellular bacteria

The requirement for iron as a critical component for cellular processes has long been appreciated. During infection with intracellular bacteria, iron is required by both the host cell and the pathogen that inhabits the host cell. Macrophages require iron as a cofactor for the execution of important antimicrobial effector mechanisms, including the NADPH dependent oxidative burst and the production of nitrogen radicals catalysed by the inducible nitric oxide synthase. On the other side of the equation, intracellular bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis have an obligate requirement for iron to support their growth and survival inside cells. This brief report summarises the background to our work on iron modulation in infections with these two organisms and highlights key observations on how modulation of host iron status disturbs the equilibrium between host and pathogen and can determine the outcome of infection.     

Contribution of phagocytosis and intracellular sensing for cytokine production by Staphylococcus aureus-activated macrophages

Toll-like receptors (TLRs) are involved in the sensing of microbially derived compounds. We analyzed the contribution of these receptors to cytokine production by macrophages following stimulation with whole bacteria. Using knockout mice, we confirmed that the TLR4 and TLR2 contribution was predominant in the induction of tumor necrosis factor alpha and interleukin-10 by gram-negative bacteria. In contrast, the absence of TLR2 and/or TLR4 or TLR9 did not affect the response to gram-positive bacteria. In the absence of TLR2, phagocytosis was essential for cytokine production in response to heat-killed Staphylococcus aureus (HKSA). Because intracellular sensing was important in the absence of TLR2, we evaluated the contribution of Nod1 and Nod2, intracytoplasmic sensors of peptidoglycan-derived muropeptides, to the response to HKSA. By transfecting RAW 264.7 macrophages with dominant negative (DN) forms of Nod1 and Nod2, we showed that both molecules inhibited NF-kappaB activation in response to HKSA. The unexpected interference of DN Nod1 in the response of macrophages to gram-positive bacteria was confirmed with a Nod2 agonist (muramyl dipeptide) in transfection experiments with HEK293T cell. Taken together, these results show the contribution of phagocytosis and Nod molecules to the response to HKSA in macrophages and also identify possible cross talk between Nod1 and Nod2.     

The use of fluorescent liposomal immunoanalysis for the diagnostics and indication of glanders and melioidosis

The authors have developed a homogenous method for the detection of the cells, surface antigen, and Ag8 of glanders and melioidosis pathogens and antibodies to them, based on the compliment-dependent lysis of Ag8-sensitized liposomes. The method is highly specific; its sensitivity to antibodies to glanders and melioidosis pathogens is 24 to 440 times higher than that of RID: its sensitivity to Ag8 is 100 ng/ml corresponding to 1.25x10-10 M, and its sensitivity to cells is 10(5) to 10(6) cells/ml).     

Function of phagocytosis and intracellular killing of peripheral neutrophils in Kawasaki disease

NBT (Nitroblue Tetrazolium) test was performed in 17 patients with Kawasaki disease to examine the function of phagocytosis and intra-cellular killing of neutrophils. The value was high compared to other pediatric patients. Activation with Proteus OX-2 antigen before NBF test showed a significant higher level than other proteus antigens, which correspond in serum level. With previous electronmicroscopic observation of rickettsia-like body in biopsy specimen, these findings suggest the existence of an agent in Kawasaki disease which shares antigenicity with Proteus OX-2.     

Monocytes in inflammatory bowel disease: phagocytosis and intracellular killing.

The ability of peripheral blood monocytes from patients with ulcerative colitis and Crohn's disease to phagocytose and kill a standard strain of Staphyloccus aureus has been studied. Using lysostaphin, a rapidly acting muralytic enzyme, phagocytosis could be accurately differentiated from intracellular killing. When compared with normal healthy individuals and patients with gastrointestinal diseases not thought to be immunologically mediated, monocytes from patients with inflammatory bowel disease showed a statistically significant increase in the number of bacteria phagocytosed in 2 hours. There was no difference, however, between patients with Crohn's disease and those with ulcerative colitis. For all groups studied, more than 95% of ingested organisms were killed, and there was no difference between groups. These results suggest that peripheral blood monocytes in patients with Crohn's disease and ulcerative colitis are activated. It is unlikely that the granulomata of Crohn's disease result from a defect in the microbicidal function of the monocyte/macrophage system.     

Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin

pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.     

The role of septins in infections with vacuole-dwelling intracellular bacteria

Septins are a relatively little understood group of GTPases that form large assemblies in cells from all eukaryotes other than plants. Septins were first identified in cell division but have also been implicated in microbial infections. Septins often associate with cytoskeletal proteins − most often described for filamentous (F-) actin − and are considered cytoskeletal components themselves. Septins have increasingly been found to partake in processes that are linked to intracellular membranes, from mitochondria to phagosomes, and evidence is accumulating that septins specifically bind to membranes. Since a number of microorganisms have specialized to live and grow inside membranous vacuoles in the cytosol of mammalian cells, this membrane-association of septins suggests that septins may also be involved in the membranous, vacuolar structures that develop around these microbes. However, data are limited on this issue: septins have been identified by proteome analysis on some microbe-bearing vacuoles, but more extensive experimental data are only available for infections with the obligate intracellular bacterium Chlamydia trachomatis. In this review article I will discuss the available data and speculate about the mechanisms of recruitment and potential functions of septins for vacuole-dwelling microorganisms, which may be peculiar to Chlamydia or may pertain more generally to this class of microbes.     

Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans

A rapid quantitative flow cytometric (FCM) assay for the combined kinetic measurement of phagocytosis and intracellular killing of fluorescein-isothiocyanate (FITC) labelled Candida albicans is described. The fraction of phagocytosing leukocytes and the numbers of attached or internalized Candida albicans per phagocytosing leukocyte were quantified by FCM, using trypan blue and a fluorescence quenching technique. Results obtained by microscopy agreed with the FCM estimates of phagocytosis. Dead, but not live, Candida albicans stained by propidium iodide (PI). Thus, both viable and intracellularly killed fungi could be discriminated and measured by FCM. Phagocyte killing determined by the FCM assay correlated with killing measured by a standard microbiological test and by methylene blue staining. The method allows rapid and accurate testing of opsonic and phagocytic functions under both experimental and clinical conditions.     

HIV-1感染患者中性粒细胞吞噬及细胞内杀菌功能的变化

AIDS是世界性传染病,近几年其发病率呈上升趋势,死亡率很高,严重威胁着人类生命和健康.这些患者大多数不是死亡于AIDS本身,而是死亡于后期的机会致病微生物感染和其他微生物的重复感染或合并感染.     

Ultrastructural visualization of intracellular porphyrin in the rat Harderian gland

The Harderian glands of male Albino rats 1-24 months of age were studied by electron microscopy. In most glands, a few acinar cells contained straight and curved trilaminar profiles identical in form to the material in the luminal masses of porphyrin pigment. They resembled the structures which several investigators have identified as crystals of protoporphyrin IX in porphyric human and mouse hepatocytes. Protoporphyrin IX is the predominant form synthesized by rat Harderian cells in vitro. The trilaminar profiles were within the cytoplasm but not within the lipid secretory vacuoles. A large number of the acinar cells had finely tubular endoplasmic reticulum (ER). However, cells containing the trilaminar forms consistently had dilated ER vesicles. This change may have been a prelude to cell death, for some pigment-containing cells attached to the acinar basal lamina also displayed fragmented organelles and a loss of density of the cytoplasmic matrix. In some acinar lumina there was abundant cell debris along with the trilaminar profiles. It is concluded that in the rat, some of the Harderian free porphyrins can be released through cell death.     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

Transfection efficiency and intracellular fate of polycation liposomes combined with protamine

Endosomal escape and nuclear entry are the two main barriers for successful non-viral gene delivery. To overcome these barriers, polyethylenimine (PEI) with a molecular weight of 800, conjugated to cholesterol (PEI 800-Chol) was synthesized to prepare polycation liposomes (PCLs). The effect of cationic polymers on transfection was investigated by pre-condensing DNA with these before using PCLs. The complexes of PCLs and protamine/DNA nanoparticles (PLPD) were introduced as efficient gene transfer vectors, and displayed obviously higher transfection efficiency (approximately 39-fold) than PCLs/DNA complexes. Kinetics of transgene expression indicated PLPD complexes could be maintained at a relatively high level over 72?h. The order of protamine addition affected the transfection of PLPD complexes. Pre-mixed and post-mixed PLPD complexes improved transfection, although the former was preferred. Distribution of FAM-labeled oligonucleotides (FAM-ODN) in cells mediated by PCLs were throughout the whole cell, while most FAM-ODN were nuclear when transfected with PLPD. These results suggest that the protonation of PEI and membrane destabilization of 1, 2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases the endosomal escape ability of vectors. The addition of protamine, containing nuclear localization signals, improved nuclear entry of DNA. The internalization pathways for PCLs involved multiple processes and were possibly dependent on cell lines.     

A radiometric assay for the combined measurement of phagocytosis and intracellular killing of Candida albicans.

A radiometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans by human polymorphonuclear leucocytes (PMNs) is presented. The assay, based upon the incorporation of 3H-uridine into the micro-organisms, makes it possible to measure phagocytosis and intracellular killing simultaneously but independently in a single sample. Thus it is possible to determine in a single assay whether increased survival of the micro-organism is due to reduced ingestion or reduced ability of the PMN to kill. The assay is objective, quantitative and convenient for clinical application. It is also suitable for analysing the effects of various agents, including serum factors and drugs, on PMN function.