Michael J. Pabst

Removal of endotoxin from protein solutions by phase separation using Triton X-114.

Endotoxin contamination of protein solutions was reduced by a phase separation technique using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice. The solution was then warmed to 37 degrees C, whereupon two phases formed. The Triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. The first cycle of phase separation produced a 1000-fold reduction of endotoxin from contaminated preparations of cytochrome c, catalase and albumin. Complete removal of endotoxin could be achieved by further cycles of phase separation. Each cycle of phase separation resulted in only a 2% loss of protein, and could be completed within 15 min. The small amount of detergent (0.018%) that persisted in protein solution could be removed by gel filtration or absorption. Proteins treated by this procedure retained normal functions. This phase separation technique provides a rapid and gentle method for removing endotoxin from protein solutions.

Invasive M1T1 group A Streptococcus undergoes a phase‐shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2‐D gel analysis of the secreted M1T1 proteome, coupled with MALDI‐TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA– M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase‐shift to a SpeB–/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte‐stimulating capacity. The proteome of the SpeB–in vivo phase‐shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage‐encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB– phase‐shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.

Lipopolysaccharide-inactivating activity of neutrophils is due to lactoferrin.

Neutrophils can inactivate lipopolysaccharide (LPS), thereby blocking the ability of LPS to prime fresh neutrophils for enhanced fMLP‐triggered release of superoxide. Here we show that inactivation of LPS by neutrophils was primarily due to lactoferrin. A time course for inactivating LPS showed that neutrophils (5 million/ml) took 30 min to inactivate 10 ng/ml LPS. Mononuclear cells could not inactivate LPS under the same conditions. Experiments with radioactive LPS showed that inactivated LPS remained in the medium and was not taken up or destroyed by the neutrophils during inactivation. Inactivated IPS still gelled Limulus lysate and primed monocytes. Cell‐free medium from neutrophil suspensions also inactivated LPS. A single LPS‐inactivating factor was purified from medium by heparin‐agarose chromatography. SDS‐PAGE showed a single band at 80 kDa, which was identified as lactoferrin by immunoblotting. Antilactoferrin immunoglobulin G removed the LPS‐inactivating activity from purified lactoferrin and cell‐free medium. Surprisingly, even purified neutrophil lactoferrin required 30 min to inactivate LPS, indicating inherently slow binding of lactoferrin to LPS J. Leukoc. Biol. 57: 865–874; 1995.

Preliminary analysis of the mouse cerebellum proteome

This paper reports on the initial analysis of protein expression in the mouse cerebellum with the proteomics approach. Proteins from cerebellar tissue homogenates were separated by two-dimensional gel electrophoresis, and the proteins were stained with colloidal Coomassie Blue to produce a high-resolution map of the cerebellum proteome. Selected proteins from this map were digested with trypsin, and the resulting tryptic peptides were analyzed by matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography-electrospray quadrupole ion trap mass spectrometry. The mass spectrometric data were used to identify the proteins through searches of the SWISSPROT protein sequence database. To date, 30 prominent proteins with various functional characteristics were identified. These data will be used for future studies of differential protein expression in mouse models of neurological disorders.

Induction of acute pancreatitis in germ-free rats: evidence of a primary role for tumor necrosis factor-alpha.

BACKGROUND Tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of the systemic manifestations associated with acute pancreatitis. The purpose of this study was to show that TNF-alpha expression in pancreatitis is a primary response and is not the result of endotoxemia. METHODS Severe acute pancreatitis was induced in germ-free rats, which have no source of endogenous endotoxin, by ductal infusion of artificial bile. Control animals underwent sham operation and ductal infusion of saline solution. TNF-alpha levels were measured by the WEHI bioassay. Endotoxin was measured by the Limulus assay. RESULTS TNF-alpha levels remained low in the sham group (mean, 24.6 +/- 8.0 pg/ml) but were significantly elevated in normal rats with pancreatitis (181 +/- 26.8 pg/ml; p < 0.001 versus sham group) and in germ-free rats with pancreatitis (213 +/- 90 pg/ml; p < 0.002 versus sham group). No endotoxin was detected in any of the experimental rats. CONCLUSIONS Our results indicate that TNF-alpha levels are elevated in acute pancreatitis despite the absence of endotoxin, indicating a primary role of TNF-alpha in this disease.

Effects of Muramyl Peptides on Macrophages, Monokines, and Sleep

Muramyl peptides are fragments of peptidoglycan from the cell walls of bacteria. Because of their unique chemistry, the immune system recognizes that muramyl peptides are products of bacteria, and it responds by becoming activated to resist infection. This resistance to infection is nonspecific, and extends to unrelated species of bacteria, fungi, and viruses. A key mechanism of the resistance to infection is activation of macrophages. Macrophage activation results in increased production of microbicidal oxygen radicals like superoxide and peroxide, and in increased secretion of inflammatory cytokines like interleukin-1β and tumor necrosis factor-α. These cytokines, besides activating neutrophils, B lymphocytes, and T lymphocytes, act on the central nervous system to induce physiological responses like fever and sleep. These physiological responses also aid in combating infection. Muramyl peptides also activate macrophages and other cells of the immune system to kill cancer cells. Muramyl peptides and similar agents will become more important as therapeutic agents in the future, due to increasing resistance of microbes to antibiotics, and increasing numbers of patients with immunodeficiencies.

An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma.

When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl‐Met‐Leu‐Phe (fMLP). The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA‐14‐PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs). We studied the mechanisms by which LA‐14‐PP or Rs‐LPS inhibited LPS‐induced responses. When neutrophils were exposed to LA‐14‐PP or Rs‐LPS for 3 min and then to Escherichia coli‐LPS, the antagonists inhibited priming for superoxide release, and also blocked up‐regulation of CD11b and adherence. This inhibition was dependent on plasma, was not overcome by higher amounts of E. coli‐LPS or plasma, and was not observed at 0°C, suggesting that E. coli‐LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists. The alternative possibility that LA‐14‐PP or Rs‐LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp). These LPS primed neutrophils in a plasma‐dependent and CD14‐dependent manner, but were not blocked by LA‐14‐PP or Rs‐LPS. When sub‐optimal concentrations of plasma were exposed to LA‐14‐PP or Rs‐LPS, and then mixed with Pg‐IPS or Bp‐LPS, followed by incubation with neutrophils, priming and up‐regulation of GD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma. Binding of LPS‐binding protein (LBP) in plasma to immobilized E. coli‐LPS was inhibited by pre‐incubation of plasma with LA‐14‐PP or Rs‐LPS. Together with the result that treatment of plasma with anti‐LBP antibody abolished the cofactor activity of plasma, these results indicated that LA‐14‐PP and Rs‐LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils. Thus LA‐14‐PP and Rs‐LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.

Proteome of monocyte priming by lipopolysaccharide, including changes in interleukin-1beta and leukocyte elastase inhibitor

BackgroundMonocytes can be primed in vitro by lipopolysaccharide (LPS) for release of cytokines, for enhanced killing of cancer cells, and for enhanced release of microbicidal oxygen radicals like superoxide and peroxide. We investigated the proteins involved in regulating priming, using 2D gel proteomics.ResultsMonocytes from 4 normal donors were cultured for 16 h in chemically defined medium in Teflon bags ± LPS and ± 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor. LPS-primed monocytes released inflammatory cytokines, and produced increased amounts of superoxide. AEBSF blocked priming for enhanced superoxide, but did not affect cytokine release, showing that AEBSF was not toxic. After staining large-format 2D gels with Sypro ruby, we compared the monocyte proteome under the four conditions for each donor. We found 30 protein spots that differed significantly in response to LPS or AEBSF, and these proteins were identified by ion trap mass spectrometry.ConclusionWe identified 19 separate proteins that changed in response to LPS or AEBSF, including ATP synthase, coagulation factor XIII, ferritin, coronin, HN ribonuclear proteins, integrin alpha IIb, pyruvate kinase, ras suppressor protein, superoxide dismutase, transketolase, tropomyosin, vimentin, and others. Interestingly, in response to LPS, precursor proteins for interleukin-1β appeared; and in response to AEBSF, there was an increase in elastase inhibitor. The increase in elastase inhibitor provides support for our hypothesis that priming requires an endogenous serine protease.

Local anesthetics inhibit priming of neutrophils by lipopolysaccharide for enhanced release of superoxide: suppression of cytochrome b558 expression by disparate mechanisms

Local anesthetics have anti‐inflammatory effects in vivo and inhibit neutrophil functions in vitro, but how these agents act on neutrophils remains unclear. Phagocytosis and bactericidal activity of neutrophils are enhanced by exposure to bacterial components such as lipopolysaccharide (LPS); this process is termed priming, which for enhanced release of superoxide (O2−) causes mobilization of intracellular granules that contain cytochrome b558, a component of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. We studied whether local anesthetics affected LPS priming for enhanced release of O2− in response to triggering by the chemotactic peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLP), and we investigated which element in the LPS signaling pathway might be the target of local anesthetics. Neutrophils were incubated with 10 ng/ml LPS and 1% plasma ± local anesthetics, washed, and triggered with fMLP. Local anesthetics all inhibited LPS priming, and 50% inhibition was at 0.1 mM tetracaine, 0.5 mM bupivacaine, 3.0 mM lidocaine, or 4.0 mM procaine. Local anesthetics inhibited LPS‐induced mobilization of specific granules and secretory vesicles. Local anesthetics inhibited LPS‐induced up‐regulation of cytochrome b558 but not LPS‐induced translocation of p47phox. Inhibition of priming by local anesthetics was reversed by washing and incubating for 5 min. Tetracaine alone, but not the other local anesthetics, inhibited LPS activation of p38 mitogen‐activated protein kinase (MAPK) and MAPK kinase 3 (kinases in the LPS signaling pathway). The p38 MAPK inhibitors SB203580 and PD169316 also blocked LPS priming. Thus, tetracaine and the other local anesthetics inhibit by disparate mechanisms, but all the local anesthetics impaired up‐regulation of cytochrome b558 and all impaired priming of NADPH oxidase by LPS.

4-(2-Aminoethyl)benzenesulfonyl fluoride attenuates tumor-necrosis-factor-α-induced blood-brain barrier opening

The effect of serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) was investigated on the prevention of tumor-necrosis-factor-alpha (TNF-alpha)-induced blood-brain barrier opening. TNF-alpha (10,000 IU) was injected intracarotidly to newborn pigs pretreated with 0, 2.4, 4.8, 9.6 and 19.2 mg/kg AEBSF (n = 6 in each group). AEBSF dose-dependently inhibited the TNF-alpha-induced increase in the blood-brain barrier permeability for sodium fluorescein (MW = 376) in all of the five brain regions examined, while only 19.2 mg/kg AEBSF could significantly (P < 0.05) decrease the change in Evans blue-albumin (MW = 67,000) transport in two regions. In conclusion, AEBSF attenuates vasogenic brain edema formation.