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...avant Speed Vac (Farmingdale, NY), and stored at -20°C until further analysis of protein binding by immunoblotting. Western blotting. Bacterial pellets were solubilized in 20-40 PI 2x SDS-PAGE buffer =-=(20)-=- and analyzed on 14% (pl5) and 12% (BPI) polyacrylamide gels. Western transfer was performed at 200 mA for 16 h in a Bio-Rad (Cambridge, MA) apparatus as described (21). Nitrocellulose blots were bloc...

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...out the structures and biological properties of cationic antimicrobial PMN proteins and peptides such as the 50-kD bactericidal! permeability-increasing protein (BPI)1 (8, 9) and the 3-4-kD defensins =-=(10)-=-. We have proposed that BPI plays a primary antibacterial role toward Gram-negative bacteria in the intact PM:N because of the very close similarity of the fate of BPIsensitive bacteria ingested by in...

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...(model 475A; Applied Biosystems, Inc., Foster City, CA). The presence in the crude acid extract of the five rabbit defensin isoforms (NP-l -5) was determined by acid-urea PAGE as described previously =-=(18)-=-. Defensins NP-1, -2, and -5 were also isolated from citric acid extracts of rabbit PMN as described previously (19). Growth inhibitory assays in artificial media. To evaluate the effect of physiologi...

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...uently, much has been learned about the structures and biological properties of cationic antimicrobial PMN proteins and peptides such as the 50-kD bactericidal! permeability-increasing protein (BPI)1 =-=(8, 9)-=- and the 3-4-kD defensins (10). We have proposed that BPI plays a primary antibacterial role toward Gram-negative bacteria in the intact PM:N because of the very close similarity of the fate of BPIsen...

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...inflammatory sites and also in the circulation during serious infections (35-37). It has been reported that PMN treated with FMLP and cytochalasin in vitro secrete large portions of their BPI content =-=(38)-=-. We have observed, under more physiologic conditions, the gradual accumu680 Levy, Ooi, Weiss, Lehrer, and Elsbach lation of substantial concentrations of BPI (- 1 ag/ml or 20 nM) and pl5s (- 15 Mug/m...

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...efensin isoforms (NP-l -5) was determined by acid-urea PAGE as described previously (18). Defensins NP-1, -2, and -5 were also isolated from citric acid extracts of rabbit PMN as described previously =-=(19)-=-. Growth inhibitory assays in artificial media. To evaluate the effect of physiologic saline on the growth inhibitory activity of granulocyte proteins, E. coli J5 (106/ml) in sodium phosphate buffer, ...

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... and the plSs actually inhibit the action of human azurocidin on E. coli (14). Moreover, studies of other antibacterial peptides of insects and amphibians (27) as well as of mammalian (pig) intestine =-=(28)-=- also do not reveal evidence of synergy. On the other hand, lactoferrin at a very high concentration (2 mg/ml) 100 CFU (%) and lysozyme (29) as well as azurocidin (100 /tg/ml; 30) or myleoperoxidase (...

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... Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/94/08/0672/11 $2.00 Volume 94, August 1994, 672-682 that consists of both oxygen-derived and nonoxidative toxic agents =-=(1, 2)-=-. Remarkable progress has been made in the identification and characterization of individual elements of both systems (3-7). By taking advantage of the availability of these proteins and peptides in p...

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...and amphibians (27) as well as of mammalian (pig) intestine (28) also do not reveal evidence of synergy. On the other hand, lactoferrin at a very high concentration (2 mg/ml) 100 CFU (%) and lysozyme =-=(29)-=- as well as azurocidin (100 /tg/ml; 30) or myleoperoxidase (31) and elastase have been shown to act synergistically toward Gram-negative bacteria. However, the potentiating effects were only observed ...

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...primary antibacterial role toward Gram-negative bacteria in the intact PM:N because of the very close similarity of the fate of BPIsensitive bacteria ingested by intact PMN or exposed to purified BPI =-=(11, 12)-=-. We recognize, however, that the coexistence in the PMN of many distinct antimicrobial entities leaves uncertain the actual contribution of individual elements to overall PMN function. We therefore i...

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... or together in whole blood against two strains of Kl-encapsulated E. coli that resist the cellular and extracellular antibacterial systems present in normal blood. As shown previously with human BPI =-=(25)-=-, in whole blood rabbit BPI potently inhibited viability of E. coli Kl/r (Fig. 9 A), an encapsulated strain producing short chain (R-chemotype) LPS that is about as sensitive as E. coli J5 to BPI in s...

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...2 nM, i.e., a reduction of about 25-fold in the BPI dose required to inhibit bacterial growth. That these interactions are truely synergistic (rather than additive) can be judged by isobolic analysis =-=(23)-=- with respect to the IC50s of BPI and pi5A (Fig. 3 C) or NP-I (Fig. 3 D) alone and in combination. NP-I and NP-2 showed similar synergistic activity with BPI, whereas NP-5 was much less active (not sh...

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... Clin. Invest. © The American Society for Clinical Investigation, Inc. 0021-9738/94/08/0672/11 $2.00 Volume 94, August 1994, 672-682 that consists of both oxygen-derived and nonoxidative toxic agents =-=(1, 2)-=-. Remarkable progress has been made in the identification and characterization of individual elements of both systems (3-7). By taking advantage of the availability of these proteins and peptides in p...

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...herefore initiated the present study, guided by what we have learned before from the interaction of the Gramnegative bacterium Escherichia coli with two granule-associated protein species of PMN, BPI =-=(13)-=-, and the 15-kD proteins (pl5s) (14, 15). We have examined the antimicrobial effect of these well-defined proteins as well as of members of the defensin family, alone and in combination. The results i...

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...primary antibacterial role toward Gram-negative bacteria in the intact PM:N because of the very close similarity of the fate of BPIsensitive bacteria ingested by intact PMN or exposed to purified BPI =-=(11, 12)-=-. We recognize, however, that the coexistence in the PMN of many distinct antimicrobial entities leaves uncertain the actual contribution of individual elements to overall PMN function. We therefore i...

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..., guided by what we have learned before from the interaction of the Gramnegative bacterium Escherichia coli with two granule-associated protein species of PMN, BPI (13), and the 15-kD proteins (pl5s) =-=(14, 15)-=-. We have examined the antimicrobial effect of these well-defined proteins as well as of members of the defensin family, alone and in combination. The results indicate not only qualitative and quantit...

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...not observed when pl5A and NP-1 are combined, and the plSs actually inhibit the action of human azurocidin on E. coli (14). Moreover, studies of other antibacterial peptides of insects and amphibians =-=(27)-=- as well as of mammalian (pig) intestine (28) also do not reveal evidence of synergy. On the other hand, lactoferrin at a very high concentration (2 mg/ml) 100 CFU (%) and lysozyme (29) as well as azu...

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...gen-induced inflammatory rabbit peritoneal exudates at molar ratios similar to those required for synergy in vitro (Weinrauch Y., et al., manuscript in preparation). Moreover, a recent clinical study =-=(39)-=- revealed that the mean normal concentration of defensins (42 ng/ml, - 12 nM) in human plasma increased dramatically during sepsis, ranging from 900 ng/ml to 180 Mg/ml (250 nM to 50 iM). Thus, the app...

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...Ss. Amino acid analysis and complete amino-terminal sequencing (not shown) revealed that the proteins in peaks 1 and 2 were the previously described antimicrobial defensins NP-1 and NP2, respectively =-=(19, 22)-=-. The less potent (and less cationic) defensins NP-3, -4, and -5 are also present in the crude extract as judged by acid-urea PAGE (not shown) but were not recovered by this affinity purification proc...

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...hat the synergism of BPI and NP-i reflects the potentiation of BPI action by NP-1. Effect on bacterial phospholipolysis. Another hallmark of BPI action is the activation of bacterial phospholipolysis =-=(24)-=-. Exposure of E. coli J5 to : 20 nM BPI or 2 5 jLM pl5A in buffered nutrient broth/physiological saline activates bacterial envelope phospholipid hydrolysis, whereas even 60 ,uM NP-i had no effect (Fi...

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...) also do not reveal evidence of synergy. On the other hand, lactoferrin at a very high concentration (2 mg/ml) 100 CFU (%) and lysozyme (29) as well as azurocidin (100 /tg/ml; 30) or myleoperoxidase =-=(31)-=- and elastase have been shown to act synergistically toward Gram-negative bacteria. However, the potentiating effects were only observed in media of low osmolarity. Activity was blocked by raising the...

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...binding may argue for potentiation of plSA action by BPI. There is a precedent for such a BPI effect: we have shown before that BPI involves another granule-associated PMN protein, a phospholipase A2 =-=(32, 33)-=-, in the destruction of target E. coli, in part by promoting binding of the enzyme to bacteria (34). On the other hand, the increase in pl5A binding per se may be insufficient to account for the incre...

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... was based on previous studies in this laboratory showing that, among the many cationic proteins and peptides in crude acid extracts of rabbit PMN, only some exhibit preferential binding to E. coli C =-=(13, 26)-=-, including the bactericidal/permeability increasing pro-U--- BPI + laM piSA ltein and the plSs (14). By varying the number of bacteria, i.e., ---G pISA alone the binding sites, during incubation with...

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...binding may argue for potentiation of plSA action by BPI. There is a precedent for such a BPI effect: we have shown before that BPI involves another granule-associated PMN protein, a phospholipase A2 =-=(32, 33)-=-, in the destruction of target E. coli, in part by promoting binding of the enzyme to bacteria (34). On the other hand, the increase in pl5A binding per se may be insufficient to account for the incre...

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... of low osmolarity. Activity was blocked by raising the osmolarity above 60 mosM and by physiologic concentrations of either Ca2+ or Mg2+ in the former case (29) or NaCl exceeding 20 mM in the latter =-=(30)-=-. In contrast, the combined effects of p15 or NP-I with BPI on E. coli are manifest in vitro in simple buffered isotonic media as well as plasma and whole blood, suggesting that these synergistic acti...

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...om the phagosome to the extracellular environment where they can also act in synergy. Recombinant BPI has been tested as a therapeutic agent against Gram-negative bacterial infections and endotoxemia =-=(40)-=-. The ability of proteins such as pISA and the more cationic defensins to act synergistically with BPI suggests that they may be used as pharmacologic adjuncts. This study sets the stage for the furth...

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..., guided by what we have learned before from the interaction of the Gramnegative bacterium Escherichia coli with two granule-associated protein species of PMN, BPI (13), and the 15-kD proteins (pl5s) =-=(14, 15)-=-. We have examined the antimicrobial effect of these well-defined proteins as well as of members of the defensin family, alone and in combination. The results indicate not only qualitative and quantit...

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...proteins and peptides in pure form, obtained either by traditional purification or by recombinant DNA methods, their antimicrobial actions as individual agents have been analyzed extensively in vitro =-=(2, 3)-=-. Consequently, much has been learned about the structures and biological properties of cationic antimicrobial PMN proteins and peptides such as the 50-kD bactericidal! permeability-increasing protein...

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...Cross (Department of Bacterial Diseases, Walter Reed Army Medical Center, Washington, DC) that were part of a collection of bacteremic isolates previously characterized by phage typing for Kl capsule =-=(16)-=-, and Staphylococcus aureus (Cowan strain) obtained from the New York University Medical School Department of Microbiology bacteria collection. Bacteria were maintained either on nutrient broth/agar s...