Preparation of rabbit alveolar macrophages in high purity and yield

Patterson-Delafield, Judith; Lehrer, Robert I.

doi:10.1016/0022-1759(80)90277-x

Cited by 10 publications

(6 citation statements)

References 5 publications

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“…In the 1980´s Lehrer [7,8] and Selsted [9] discovered and characterized defensins from rabbit lung macrophages (for a recent review on defensins see Ganz [10]), and Zasloff's [11] discovered antibacterial magainin peptides from the skin of the African clawed frog Xenopus laevis. Antibacterial peptides occur widespread in nature, and so far antibacterial peptides have been found in all organisms investigated [12].…”

Section: Introductionmentioning

confidence: 99%

The Medicinal Chemistry of Short Lactoferricin-Based Antibacterial Peptides

Haug

Strøm

Svendsen

2007

CMC

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This review discusses antibacterial peptides from the perspective of development into clinically useful chemotherapeutic drugs using short lactoferricin based peptides as examples. The review shows how important features for antibacterial activity can be identified and explored using the molecular properties of a range of natural and non-natural amino acids. The results have been further refined quantitatively using a "soft-modelling" approach where important structural parameters that influence the antibacterial activity of 15-residue model peptides were identified. The review describes how this knowledge is utilised to generate pharmacophores for antibacterial efficacy. These pharmacophores turn out to be surprisingly small and relatively consistent between typical Gram-negative and Gram-positive bacteria leading to the discovery of a novel class of short synthetic cationic antimicrobial peptides. These compounds are found to have high antibacterial activity against several bacterial strains that are resistant to commercial antibiotics, and are promising as future clinical candidates for treatment of infections caused by several clinically relevant pathogens.

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Section: Introductionmentioning

confidence: 99%

The Medicinal Chemistry of Short Lactoferricin-Based Antibacterial Peptides

Haug

Strøm

Svendsen

2007

CMC

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“…They are composed of 29-42 amino acids and contain three disulphide bonds, formed by three pairs of cysteine residues [1][2][3][4]. Defensins have been isolated from various tissue sources, such as mammalian trachea, intestine, tongue and skin, and human plasma, oral mucosa, salivary glands, saliva and ocular surface [5][6][7][8][9][10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Large-scale synthesis and functional elements for the antimicrobial activity of defensins

Raj

Antonyraj

Karunakaran

2000

Biochemical Journal

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Human neutrophil defensins, and their analogues incorporating anionic, hydrophobic or cationic residues at the N- and C-termini, were synthesized by solid-phase procedures. The synthetic defensins were examined for their microbicidal activity against Candida albicans, two Gram-negative bacteria (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) and two Gram-positive bacteria (Streptococcus gordonii and Streptococcus mutans). The human neutrophil peptide 1 (HNP1) and HNP2 were found to be potent candidacidal agents. HNP3, which differs by one amino acid at the N-terminus of its sequence, was totally inactive. The Gram-negative bacteria A. actinomycetemcomitans and P. gingivalis and the Gram-positive bacteria S. gordonii and S. mutans were insensitive to human defensins. However, the insertion of two basic residues, such as arginine, at both the N-terminus and the C-terminus of HNP2 significantly enhanced antifungal and antibacterial activity. The addition of anionic residues, such as aspartic acid, at the N- and C-termini rendered the molecule totally inactive. The presence of two hydrophobic amino acids, such as valine, at the N-terminus of HNP2 and of two basic arginine residues at its C-terminus resulted in molecules that were optimally active against these oral pathogens. The results suggest that the N- and C-terminal residues in defensin peptides are the crucial functional elements that determine their microbicidal potency. The three-dimensional structure of all defensins constitutes the same amphiphilic β-sheet structure, with the polar face formed by the N- and C-terminal residues playing an important role in defining microbicidal potency and the antimicrobial spectrum. The enhanced microbicidal activity observed for defensin peptides with two basic residues at both the N- and C-termini could be due to optimization of the amphiphilicity of the structure, which could facilitate specific interactions with the microbial membranes.

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“…For these experiments, rabbit lung macrophages were used because the lungs provide an in vivo source of a relatively pure (> 95%) preparation of macrophages. 16 Moreover, these cells have been used previously for lipoprotein uptake studies. 24 The utilization of PMV labeled with 125 I by macrophages was assessed by the release of radioactive trichloroacetic acidsoluble degradation products into the incubation medium.…”

Section: Resultsmentioning

confidence: 99%

“…Alveolar macrophages were obtained by lavage with PBS containing 4.5 mM glucose and heparin (1,000 U/ml) by the method of Patterson-Delafield and Lehrer. 16 The lavage was centrifuged at 500 g for 10 minutes and the cells were suspended and sedimented in PBS diluted with water (1:5) to lyse the contaminating erythrocytes. This procedure yields an essentially pure (>95%) population of macrophages.…”

Section: Isolation Of Alveolar Macrophagesmentioning

confidence: 99%

“…This procedure yields an essentially pure (>95%) population of macrophages. 16 Macrophages (5 x 10 s cells) were added to 0.5 ml of nutritionally complete Dulbecco modified Eagle's medium (DMEM) supplemented with nonessential and essential amino acids, 4.7 mM NaHCO 3 ,10% fetal calf serum, and a mixture of antibiotics (streptomycin, penicillin, garamycin, and amphotericin). After a 2-hour incubation at 37° C, adhering cells were washed with fresh PBS and maintained in nutritionally complete DMEM under a humidified atmosphere of 95% Oj/5% CO 2 at 37° C.…”

Section: Isolation Of Alveolar Macrophagesmentioning

confidence: 99%

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Accumulation of cholesteryl ester and lipid droplets in macrophages after uptake of cholesterol-rich necrotic products.

Wong¹,

Hashimoto²

1987

Arteriosclerosis

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The prevalence of degenerated cells in the thickened intima of cholesterol-fed animals suggested that necrotic regions might become a focus for lesion development by attracting histiocytes or monocytes. We propose that cholesterol-rich necrotic products scavenged by macrophages incite the accumulation of cholesteryl ester and the formation of cholesteryl ester droplets. The feasibility of this hypothesis was tested with readily available materials, namely, lung macrophages and plasma membrane vesicles (PMV) released from dying cholesterol-rich and normal hepatocytes. PMV from cholesterol-rich hepatocytes contained 30-fold more cholesteryl ester than PMV from normal hepatocytes. Degradation of 125 I-PMV to trichloroacetic acid-soluble products by macrophages was fourfold higher than that of 125 l-acetylated low density lipoprotein (acetyl-LDL) at 100 /xg/ml of ligand in the incubation medium. Within this concentration range, degradation of PMV increased almost linearly with increasing concentrations of PMV, while the degradation of acetyl-LDL followed a saturation curve. Cholesterol-rich PMV increased the cholesteryl ester content of macrophages fourfold and augmented the incorporation of oleate threefold relative to normal PMV. These studies were extended to aortic smooth muscle cells. As with hepatocyte-PMV, smooth muscle cell-PMV was internalized and degraded by macrophages T here is little doubt that abnormally high concentrations of low density lipoprotein (LDL) in plasma incite atherogenesis and that lipoprotein cholesterol accumulates in atherosclerotic lesions as cholesteryl ester droplets in macrophages. 1 The events in the formation of atherosclerotic lesions are understood in outline only. Cell culture studies have indicated that modification of LDL either by chemical reagents 2 " 4 or by conditioning with endothelial cells 5 is necessary to stimulate the accumulation of cholesteryl ester in macrophages, implicating abnormal instead of native LDL in atherogenesis. Indeed, modified LDL isolated from lesions abet in vitro accumulation of macrophage cholesteryl ester.6 Low density lipoprotein modified by incubation with endothelial cells also exhibit cytotoxic properties.7 The latter may also play a role in atherogenesis.We have considered an alternative pathway for the formation of cholesteryl ester-rich macrophages which may or may not involve modified LDL. Prevalence of necrotic cells in thickened intima of hypercholesterolemic swine 8 ' 9 suggested that necrotic products may become another source of cholesterol for infiltrated macrophages over and above that provided by modified LDL. A common response of several cell types to injury and the dying process is the formation of blebs on the cell surface which subsequently pinch off to form plasma membrane vesicles. 10 " 12 These vesicles are enriched with cholesterol and could conceivably be enriched even more if derived from a parent cholesterol-rich cell. We propose that plasma membrane vesicles (PMV) released from dying cells in the thickened inti...

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Preparation of rabbit alveolar macrophages in high purity and yield

Cited by 10 publications

References 5 publications

The Medicinal Chemistry of Short Lactoferricin-Based Antibacterial Peptides

The Medicinal Chemistry of Short Lactoferricin-Based Antibacterial Peptides

Large-scale synthesis and functional elements for the antimicrobial activity of defensins

Accumulation of cholesteryl ester and lipid droplets in macrophages after uptake of cholesterol-rich necrotic products.

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