Nile red: a selective fluorescent stain for intracellular lipid droplets.

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

Enhancement of resistance to coccidiosis and necrotic enteritis in broiler chickens by dietary muscadine pomace.

Muscadine pomace (MP), a by-product of the production of wine and juice from Vitis rotundifolia, was dried and tested in chickens for effects on primary resistance to coccidiosis, development of protective immunity after vaccination with live coccidia, and resistance to necrotic enteritis (NE) caused by the joint action of Clostridium perfringens and coccidia. To test primary resistance to coccidiosis, 2-wk-old chicks were given 2% or 5% MP in the diet and inoculated with Eimeria acervulina and E. maxima. Birds given MP at either level had significantly (P < 0.05) lower lesion scores at 7 days postinoculation, in comparison with control birds, although weight gains were statistically similar. Broiler chickens were given 2% or 5% MP and grown to 42 days to test the palatability of MP. Birds given 2% MP in feed grew similarly to untreated controls, but birds given 5% had poorer average live weight. This suggested a negative effect on feed intake at the higher level. The effects of dietary 0.5% or 2.0% MP on immune protection were tested after live coccidiosis vaccination in the hatchery. Chicks were removed from each pen at 21 days of age and challenged with E acervulina, E. maxima, and E. tenella. Resistance to infection was improved by MP as suggested by significantly (P < 0.05) lower lesion scores 7 days postchallenge, and improved weight gains in comparison with immunized control birds that did not receive MP. At 42 days of age, birds given MP had higher average live weights than controls, although feed efficiency was not affected. An established model was used to study the effect of MP on NE in broiler chickens. Chicks were inoculated with live coccidia at 14 days of age and dosed orally with live cultures of C perfringens on day 19, day 20, and day 21. Enteritis caused 48% mortality in the first study and 67% mortality in the second study. Dietary MP at 0.5-2.0% significantly (P < 0.05) reduced mortality in both experiments; improved weight gain relative to the unmedicated, infected control; and reduced lesion scores at necropsy. Overall, the results of six experiments suggested that MP given in the diet at 0.5% or higher had a positive effect on primary resistance and development of acquired resistance to two severe intestinal diseases in chickens.

Phosphatidylserine-mediated delivery of cholesterol to macrophages: a novel experimental method for the generation of foam cells.

P388D1 macrophages were incubated for 24 h with cholesterol-phosphatidylserine liposomes (50 micrograms cholesterol/ml) and the content of cellular cholesteryl esters increased to approx. 200 micrograms/mg cell protein. Similar results were not observed with cholesterol-phosphatidylcholine liposomes. These results demonstrate that specific phospholipid-cholesterol liposomes can be utilized for the experimental production of macrophage cholesterol-rich foam cells.

Association of negatively-charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.

LDL, the major carrier of cholesterol in blood, is poorly metabolized by macrophages. In contrast, macrophages can recognize and endocytose anionic phospholipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin. Since macrophages can take up large amounts of these phospholipids, experiments were performed to ascertain whether pre-incubation of native LDL with negatively-charged phospholipids would enhance the metabolism of LDL by macrophages. When 125I-LDL was incubated with cardiolipin liposomes for 18 h at 37 degrees C before addition to macrophages, an approx. 40-fold increase of LDL metabolism by these cells was observed. Similar results were found when LDL was pre-incubated with phosphatidylserine or phosphatidylglycerol; however, pre-incubation of LDL with phosphatidylcholine liposomes did not lead to an increase of LDL metabolism. The macrophage uptake of LDL pre-incubated with cardiolipin was reduced to approx. 40% of control values in the presence of dextran sulfate and fucoidin, inhibitors of anionic phospholipid uptake. Cytochalasin D, an inhibitor of phagocytosis, reduced the lysosomal degradation of LDL pre-incubated with cardiolipin to approx. 10% of control values. When the LDL-cardiolipin mixture was chromatographed on agarose gel, two peaks containing LDL were observed in the elution profile: the first peak appeared at the void volume and the second peak was detected just ahead of native LDL. The LDL in both peaks was much more extensively metabolized by macrophages than was native LDL; the LDL in the first peak was metabolized at a rate that was 8 times the second peak. The results demonstrate that negatively-charged phospholipids can form a complex with LDL which facilitates its phagocytosis by macrophages.

Immunohistochemical studies on electron transport proteins associated with cytochromes P-450 in steroidogenic tissues. II. Microsomal NADPH-cytochrome c reductase in the rat adrenal.

NADPH-cytochrome c reductase (NADPH : ferricytochrome oxido-reductase, EC 1.6.2.4), the flavoprotein which mediates the NADPH-dependent reduction of cytochromes P-450 in adrenocortical microsomes, has been localized immunohistochemically at the light microscopic level in rat adrenal glands. Localization was achieved through the use of sheep antiserum produced against purified, trypsin-solubilized rat hepatic microsomal NADPH-cytochrome c reductase in both an unlabeled antibody peroxidase-antiperoxidase technique and an indirect fluorescent antibody method. The sheep antibody to rat hepatic microsomal NADPH-cytochrome c reductase concomitantly inhibited the NADPH-cytochrome c reductase and progesterone 21-hydroxylase activities catalyzed by isolated rat adrenal microsomes. When sections of rat adrenal glands were exposed to the reductase antiserum in both immunohistochemical procedures, positive staining for NADPH-cytochrome c reductase was observed in parenchymal cells of the three cortical zones but not in medullary chromaffin cells. The intensity of staining, however, was found to differ among the three cortical zones, with the most intense staining being found in the zona fasciculata and the least in the zona glomerulosa. The intensity of staining was also found to differ among cells within the zona fasciculata. These immunohistochemical observations demonstrate that microsomal NADPH-cytochrome c reductase is not distributed uniformly throughout the rat adrenal cortex.

Iron-ascorbate-phospholipid mediated modification of low density lipoprotein.

LDL can be oxidized by a variety of agents to form a modified lipoprotein which is capable of being avidly metabolized by macrophages. While previous in vitro studies have focused exclusively on the oxidation of LDL, other lipids found in the atheroma are also subject to oxidation and its lipoperoxide byproducts may contribute to the process of LDL modification. To examine the relationship between the oxidation of phospholipids and the subsequent modification of LDL, we incubated 250 microM phosphatidylcholine with 10 microM ferrous sulfate and 50 microM ascorbic acid in 10 mM Tris (pH 7.0). After 18 h at 37 degrees C, significant amounts of thiobarbituric acid reactive substances (TBARS) were formed. The inclusion of LDL (100 micrograms protein/ml) elevated the TBARS and increased the electrophoretic mobility of the lipoprotein. LDL treated with iron and ascorbate in the absence of phosphatidylcholine did not result in the modification of this lipoprotein. LDL that was incubated with phosphatidylcholine, iron and ascorbate was found to be metabolized by macrophages to a far greater extent than native LDL or LDL treated with phosphatidylcholine alone. Probucol (10 microM) inhibited the LDL modification process. These results demonstrate that while iron and ascorbate cannot oxidize LDL directly, the addition of phosphatidylcholine to these initiators of lipid peroxidation can mediate and lead to the modification of LDL.

Endocytosis of sulfatides by macrophages: relationship to the cellular uptake of phosphatidylserine.

These studies initially examined the effect of sulfatides on the endocytosis of phosphatidylserine (PS) liposomes in J774 macrophages employing liposomes composed entirely of PS and the nonexchangeable radiolabel [3H]cholesteryl hexadecyl ether. Bovine brain sulfatides significantly inhibited the uptake of PS liposomes by macrophages to a level of approximately 15% of control values. To examine whether macrophages were also capable of recognizing and internalizing sulfatides, sulfatide-containing liposomes were prepared using phosphatidylcholine (PC), cholesterol, and sulfatides (6:2:4 molar ratio) incorporating the same radiolabel. The sulfatide-containing liposomes were found to be avidly endocytosed by macrophages. Uptake of the sulfatide-containing liposomes by macrophages was significantly greater than the uptake of liposomes made without sulfatides. When the macrophages were incubated with the anionic compounds dextran sulfate and fucoidin, both the binding of the liposomes to the macrophages at 4 degrees C and the internalization of the liposomes at 37 degrees C were inhibited to approximately 10% of control values. The negatively charged phospholipids phosphatidylglycerol and cardiolipin significantly inhibited the uptake of sulfatide-containing liposomes, and PS was not effective in reducing the cellular uptake of these liposomes. Both oxidized low-density lipoprotein (LDL) and acetylated LDL reduced the uptake of the sulfatide-containing liposomes; the uptake observed in the presence of acetylated LDL and oxidized LDL was approximately 70% and 40%, respectively, of control values. These findings demonstrate that macrophages can efficiently endocytose both sulfatides and negatively charged phospholipids to remove them from the circulation.

Cholesteryl esterase-treated LDL augments oxidized LDL-mediated cholesteryl ester deposition in mouse peritoneal macrophages.

Arterial unesterified cholesterol, phospholipid particles have been isolated from atherosclerotic lesions and characterized. However, the role of these 'liposomes' in macrophage foam cell formation is unclear. Recently, LDL, after trypsin and cholesteryl esterase treatment (T/CE LDL), was shown to have physical properties similar to the unesterified cholesterol, phospholipid particles isolated from atherosclerotic lesions. Yet, when mouse peritoneal macrophages were incubated with these model particles in culture medium (DMEM and 5% LPDS), only an insignificant accumulation of cellular cholesteryl esters was observed. Previously, we demonstrated that complex formation between unesterified cholesterol, phosphatidylcholine liposomes and cupric sulfate-oxidized LDL dramatically enhances the ability of the liposomes to augment cellular cholesterol accretion (Greenspan P, Yu H, Mao F, Gutman RL. J Lipid Res 1997;38:101-109). When T/CE LDL, another cholesterol-rich phospholipid particle, was substituted for unesterified cholesterol phosphatidylcholine liposomes in our complex, mouse peritoneal macrophages accumulated a significant amount of both cellular unesterifed cholesterol (61 microg/mg cell protein) and cholesteryl esters (76 microg/mg cell protein) after 48 h of incubation. These results demonstrate again that the interaction of two cholesterol-bearing particles (T/CE LDL and oxidized LDL), which individually can not promote significant cholesterol accumulation in cells, will, when combined, produce macrophage foam cells.

Carboxy-substituted cinnamides: a novel series of potent, orally active LTB4 receptor antagonists.

A series of carboxy-substituted cinnamides were investigated as antagonists of the human cell surface leukotriene B4 (LTB4) receptor. Binding was determined through measurement of [3H]LTB4 displacement from human neutrophils. Receptor antagonism was confirmed through a functional assay, which measures inhibition of Ca2+ release in human neutrophils. Potent antagonists were discovered through optimization of a random screening hit, a p-(alpha-methylbenzyloxy)cinnamide, having low-micromolar activity. Substantial improvement of in vitro potency was realized by the attachment of a carboxylic acid moiety to the cinnamide phenyl ring through a flexible tether, leading to identification of compounds with low-nanomolar potency. Modification of the benzyloxy substituent, either through ortho-substitution on the benzyloxy phenyl group or through replacement of the ether oxygen with a methylene or sulfur atom, produced achiral antagonists of equal or greater potency. The most potent compounds in vitro were assayed for oral activity using the arachidonic acid-induced mouse ear edema model of inflammation. Several compounds in this series were found to significantly inhibit edema formation and myeloperoxidase activity in this model up to 17 h after oral administration. Representatives of this series have been shown to be potent and long-acting orally active inhibitors of the LTB4 receptor.

Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design.

Cathepsin B is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. As part of an effort to identify potent, reversible inhibitors of this protease, we examined a series of dipeptidyl nitriles, starting with the previously reported Cbz-Phe-NH-CH(2)CN (19, IC(50) = 62 microM). High-resolution X-ray crystallographic data and molecular modeling were used to optimize the P(1), P(2), and P(3) substituents of this template. Cathepsin B is unique in its class in that it contains a carboxylate recognition site in the S(2)' pocket of the active site. Inhibitor potency and selectivity were enhanced by tethering a carboxylate functionality from the carbon alpha to the nitrile to interact with this region of the enzyme. This resulted in the identification of compound 10, a 7 nM inhibitor of cathepsin B, with excellent selectivity over other cysteine cathepsins.

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.

Does quality of care affect rates of hospitalization for childhood asthma?

BACKGROUND: Hospitalization rates for childhood asthma are three times as high in Boston, Massachusetts, as in Rochester, New York; New Haven, Connecticut, rates are intermediate. We undertook this study to determine how care for children admitted for asthma varies across these communities. METHODS: We performed a community-wide retrospective chart review. We reviewed a random sample of all asthma hospitalizations, from 1988 to 1990, of children 2 to 12 years old living in these communities (n = 614). Abstracted data included demographics, illness severity, and treatment before admission. RESULTS: Compared with Rochester children, Boston children were less likely to have received maintenance preventive therapy (inhaled corticosteroids or cromolyn [odds ratio (OR), 0.4 (0.2, 0.9)]), acute "rescue" therapy (oral corticosteroids [OR, 0.2 (0.1, 0.4)]), or inhaled beta-agonist therapy [OR, 0.5 (0.3, 1.0)]. A larger proportion of admitted asthmatic patients in Boston (34%) were in the least severely ill group-oxygen saturation 95% or above-compared with patients in Rochester (20%). CONCLUSIONS: The quality of ambulatory care, including choice of preventive therapies and thresholds for admission, likely plays a key role in determining community hospitalization rates for chronic conditions such as childhood asthma.

Cellular localization of antiviral polyoxometalates in J774 macrophages.

The cellular localization of the polyoxometalates, K12H2[P2W12O48].24H20 (JM 1591), K10[P2W18-Zn4(H2O)2O68].20H2O (JM 1596), and [Me3NH]8[Si2W18Nb6O77] (JM 2820) were examined in cultured J774 cells by inhibition of cellular uptake of acetylated low-density lipoprotein (LDL) and by electron microscopy. All three polyoxometalates inhibited the cellular uptake of acetylated LDL, suggesting that the polyoxometalates block the association of acetylated LDL with cellular scavenger receptors. Fluorescence microscopy showed increased numbers of vacuoles in the presence of polyoxometalates, suggesting their uptake by cells. Using scanning electron microscopy (SEM), no significant cell surface morphological differences were observed between treated and non-treated J774 cells, suggesting that the compounds are not toxic to J774 cells up to a concentration of 200 micrograms/ml. Transmission electron microscopy (TEM) revealed large amounts of high electron dense granules were observed in the ramifying system of tubular cavities and vacuoles. TEM-energy dispersive spectroscopy (EDS) X-ray microanalysis was unable to differentiate the dense particles, most likely because the amount of tungsten in the cells was below the limit of detection. X-ray microanalysis conducted using the SEM-wavelength dispersive spectroscopy (WDS) detected tungsten, averaging 0.45 +/- 0.16% (mean +/- S.D.), in the J774 cells treated with JM 2820, suggesting that this polyoxometalate was taken up by the macrophages or was bound to their surface. Polyoxometalates interact at the cell surface and appear to be taken up by J774 macrophages. The cellular localization of polyoxometalates may be associated with anti-HIV activity.

Primary care involvement among hospitalized children.

OBJECTIVE: To examine relations between characteristics of a child's usual source of primary care and involvement of that source before and during hospitalization. DESIGN: Medical record review of pediatric hospitalizations. SETTING: All hospitals in Boston, Mass; New Haven, Conn; and Rochester, NY admitting children during the calendar years 1988 through 1990. PATIENTS: The study included 1875 randomly selected pediatric hospitalizations for five diagnostic groups (i.e., asthma and other lower respiratory tract disease, abdominal pain [including appendicitis], meningitis [bacterial and viral], toxic ingestions, and head injury). Hospital records selected were limited to children aged between 1 month and 12 years and residing in the three study communities. OUTCOME MEASURES: Whether the primary care source examined the child before admission to the hospital, referred the child to the emergency department, or served as the in-hospital attending physician. RESULTS: Of the medical charts reviewed, 85.7% identified primary care sources. Children in Rochester had higher rates of medical visits before admission (P < .04), referrals (P < .001), and in-hospital care provided by the primary care physician (P < .001, chi 2) than children in Boston and New Haven. Patterns of primary care involvement also varied by source of care within cities, after controlling for income and severity of illness. Compared with children from Rochester community-based private practices, children in Boston receiving care from health centers, hospitals, or community-based private practices generally had 25% to 50% lower likelihood of positive findings on all primary care involvement measures. Children in New Haven receiving care from community-based private or hospital-based practices also had lower rates, but involvement rates were not higher when they received care from health centers. Other children in Rochester and children receiving care from health maintenance organizations in all cities demonstrated almost no significant differences compared with data from Rochester community practices. CONCLUSION: The source of primary care is associated with patterns of prehospital and hospital care among hospitalized children, although specific associations vary by city.

LTB4-induced transient neutropenia in the rat: a model for evaluating efficacy and bioavailability of LTB4 receptor antagonists.

An animal model of leukotriene B4- (LTB4) induced neutropenia has been developed to evaluate LTB4 receptor antagonists in vivo. LTB4, a potent chemotactic inflammatory mediator, when administered intravenously, induces a profound, rapid, and transient redistribution of blood neutrophils from the circulating pool to the marginated pool. This phenomenon is applied in the neutropenia model whereby circulating blood neutrophil counts prior to and after intravenous infusion of LTB4 are compared. Kinetics of LTB4-induced neutrophil responses are determined through the use of a Technicon H*1 automated blood cell analyzer. LTB4 receptor antagonists are identified by inhibition of LTB4-induced neutropenia. Standard antiinflammatory compounds including BW-755C, Abbott A-64077 (zileuton), dexamethasone-21-acetate, indomethacin, and naproxen did not affect LTB4-induced neutropenia. A potent LTB4 receptor antagonist, designated "RPR," inhibited LTB4-induced neutropenia following oral administration in a dose-dependent fashion.

Advances in agarose gel electrophoresis of serum lipoproteins.

Agarose gel electrophoresis has been extensively employed by researchers to gain a greater understanding of lipoprotein biology and its relationship to cardiovascular disease. Advances in this technique have been made in the visualization and quantitation of separated lipoproteins, in the use of agarose gel electrophoresis for detection and quantitation of apolipoproteins of the separated lipoproteins, and in the detection of lipoprotein heterogeneity. Agarose gel electrophoresis has been employed for two-dimensional electrophoretic analysis of lipoproteins as well as in several different methods which probe the immunological properties of lipoproteins. Agarose gel electrophoresis has thus become an important tool in the study of serum lipoproteins in both clinical and basic science laboratories.

Use of Nile red stain in the detection of cholesteryl ester accumulation in acid lipase-deficient fibroblasts.

The fluorescent hydrophobic probe Nile red was used to distinguish between normal human fibroblasts and fibroblasts from individuals with a genetic deficiency in lysosomal acid lipase activity (Wolman's disease and cholesteryl ester storage disease). The fluorescence of Nile red-stained cultured mutant cells, indicative of neutral lipid accumulation, was intense when compared microscopically with normal fibroblasts. The cholesteryl ester accumulation in the acid lipase-deficient fibroblasts was demonstrated qualitatively and quantitatively when cellular lipid extracts were subjected to thin-layer chromatography, followed by Nile red plate treatment and fluorescence spectrometry scanning. These results demonstrate the utility of the Nile red stain to document cellular lipid overloading. The techniques are simple to perform and can effectively supplement the standard enzymatic analysis used in the diagnosis of acid lipase deficiency.