scholarly journals Effects of Amoxicillin, Gentamicin, and Moxifloxacin on the Hemolytic Activity of Staphylococcus aureus In Vitro and In Vivo

2001 ◽  
Vol 45 (1) ◽  
pp. 196-202 ◽  
Author(s):  
Dieter Worlitzsch ◽  
Hayal Kaygin ◽  
Andrea Steinhuber ◽  
Axel Dalhoff ◽  
Konrad Botzenhart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Neutrophil Elastase ◽  
Human Neutrophil Elastase ◽  
Mssa Strain ◽  
Subinhibitory Concentrations ◽  
High Level ◽  
Strain Dependent ◽  
Negative Mutant

ABSTRACT In Staphylococcus aureus infection hemolysis caused by the extracellular protein α-toxin encoded by hla is thought to contribute significantly to its multifactorial virulence. In vitro, subinhibitory concentrations of β-lactam antibiotics and fluoroquinolones increase the levels of hla and α-toxin expression, whereas aminoglycosides decrease the levels ofhla and α-toxin expression. In the present study we investigated the effects of subinhibitory concentrations of amoxicillin, gentamicin, and moxifloxacin on hla and α-toxin expression and total hemolysis of S. aureusstrain 8325-4, a high-level α-toxin producer, and its α-toxin-negative mutant, DU 1090, in vitro and in a rat model of chronic S. aureus infection. The levels of expression ofhla and α-toxin and total hemolysis did not differ significantly when amoxicillin, gentamicin, or moxifloxacin was added to cultures of S. aureus strain 8325-4. In vivo, strain 8325-4 induced a significantly increased level of hemolysis in infected pouches compared to that in uninfected control pouches, but the hemolysis was reduced to control levels by treatment with doses of amoxicillin, gentamicin, or moxifloxacin that reduced bacterial numbers by 2 orders of magnitude. Additionally, the effects of subinhibitory concentrations of the three antibiotics on total hemolysis of four methicillin-resistant S. aureus and three methicillin-sensitive S. aureus (MSSA) clinical isolates were assessed in vitro. A significant increase in total hemolysis was observed for only one MSSA strain when it was treated with amoxicillin but not when it was treated with moxifloxacin or gentamicin. When purified α-toxin was incubated with purified human neutrophil elastase, α-toxin was cleaved nearly completely. The results suggest that the penicillin-induced increases in S. aureusα-toxin expression are strain dependent, that reduction of bacterial numbers in vivo counteracts this phenomenon effectively, and finally, that in localized S. aureus infections α-toxin activity is controlled by neutrophil elastase.

Peptidyl .alpha.-ketoheterocyclic inhibitors of human neutrophil elastase. 3. In vitro and in vivo potency of a series of peptidyl .alpha.-ketobenzoxazoles

1995 ◽  
Vol 38 (20) ◽  
pp. 3972-3982 ◽  
Author(s):  
Philip D. Edwards ◽  
Mark A. Zottola ◽  
Matthew Davis ◽  
Joseph Williams ◽  
Paul A. Tuthill
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Human Neutrophil Elastase

scholarly journals Novel and Modified Neutrophil Elastase Inhibitor Loaded in Topical Formulations for Psoriasis Management

Pharmaceutics ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 358 ◽  
Author(s):  
Andreia Nunes ◽  
Joana Marto ◽  
Lídia Maria Gonçalves ◽  
Sandra Simões ◽  
Rita Félix ◽  
...  
Keyword(s):  
Serine Protease ◽  
Neutrophil Elastase ◽  
In Vitro Cytotoxicity ◽  
Human Neutrophil Elastase ◽  
Therapeutic Effects ◽  
Neutrophil Elastase Inhibitor ◽  
Analytical Methodology ◽  
Elastase Inhibitor

Human neutrophil elastase (HNE) is a serine protease that degrades matrix proteins. An excess of HNE may trigger several pathological conditions, such as psoriasis. In this work, we aimed to synthesize, characterize and formulate new HNE inhibitors with a 4-oxo-β-lactam scaffold with less toxicity, as well as therapeutic index in a psoriasis context. HNE inhibitors with 4-oxo-β-lactam scaffolds were synthesized and characterized by NMR, FTIR, melting point, mass spectrometry and elemental analysis. In vitro cytotoxicity and serine protease assays were performed. The compound with the highest cell viability (AAN-16) was selected to be incorporated in an emulsion (AAN-16 E) and in a microemulsion (AAN-16 ME). Formulations were characterized in terms of organoleptic properties, pH, rheology, droplet size distribution, in vitro drug release and in vivo psoriatic activity. All compounds were successfully synthesized according to analytical methodology, with good yields. Both formulations presented suitable physicochemical properties. AAN-16 E presented the most promising therapeutic effects in a murine model of psoriasis. Overall, new HNE inhibitors were synthesized with high and selective activity and incorporated into topical emulsions with potential to treat psoriasis.

Oxidation-resistant variants of recombinant anti-leucoprotease are better inhibitors of human-neutrophil-elastase-induced emphysema in hamsters than natural recombinant antileucoprotease

1991 ◽  
Vol 81 (6) ◽  
pp. 777-784 ◽  
Author(s):  
A. Rudolphus ◽  
R. Heinzel-Wieland ◽  
V. A. M. M. Vincent ◽  
D. Saunders ◽  
G. J. Steffens ◽  
...  
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Site Directed Mutagenesis ◽  
Human Neutrophil Elastase ◽  
Polymorphonuclear Leucocytes ◽  
Inhibitory Effects ◽  
Oxidative Inactivation ◽  
Methionine Residues

1. Antileucoprotease, being sensitive to oxidative inactivation, can be produced by recombinant techniques. Via site-directed mutagenesis, two mutants of recombinant antileucoprotease were produced in which one or more of the oxidation-sensitive methionine residues were replaced by leucine: in rALP242, methionine-73 was replaced by leucine, and in rALP231, leucine was substituted for four methionine residues. In vitro, native antileucoprotease and the recombinant antileucoprotease preparations have similar inhibitory characteristics towards human neutrophil elastase. We hypothesized that replacement of methionine residues in the antileucoprotease molecule would result in a reduced oxidation sensitivity of the mutants. 2. After incubation of recombinant antileucoprotease and its mutants with increasing dosages of cis-platinum(II)diammine dichloride, we observed that native antileucoprotease and recombinant antileucoprotease were inactivated by this reagent to the same extent. Compared with this, rALP242 was less inactivated, whereas the inhibitory capacity of rALP231 was not influenced by cis-platinum(II)diammine dichloride at all. 3. After incubation of recombinant antileucoprotease, rALP242 and rALP231 with triggered polymorphonuclear leucocytes, which are thought to produce an excess of oxidants, we measured residual inhibitory activities towards human neutrophil elastase of 10%, 55% and 87%, respectively. 4. In vivo, the inhibitory effects of intratracheally administered rALP242 and rALP231 towards human-neutrophil-elastase-induced emphysema were significantly greater than that of recombinant antileucoprotease. There were no significant differences between the mutants. With respect to secretory cell metaplasia and haemorrhage, rALP231 tended to be a better inhibitor than recombinant antileucoprotease and rALP242. 5. We conclude that the recombinant antileucoprotease mutants are less sensitive to oxidation and consequently inhibit human-neutrophil-elastase-induced emphysema to a greater extent than recombinant antileucoprotease.

Scutellarin Attenuates Human-Neutrophil-Elastase-Induced Mucus Production by Inhibiting the PKC-ERK Signaling Pathway in Vitro and in Vivo

2011 ◽  
Vol 39 (06) ◽  
pp. 1193-1206 ◽  
Author(s):  
De-Peng Jiang ◽  
Qi Li ◽  
Jie Yang ◽  
Juliy M. Perelman ◽  
Victor P. Kolosov ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Erk Signaling ◽  
Control Group ◽  
Human Neutrophil Elastase ◽  
Dependent Manner ◽  
Mucus Production

The aim of this study was to investigate the influence of scutellarin on mucus production induced by human neutrophil elastase (HNE) and the possible in vitro and in vivo mechanisms. To this purpose, cells were incubated with saline, scutellarin or gefitinib for 60 min and exposed to 0.1 μM HNE for 24 h. After being pretreated respectively with saline, scutellarin or gefitinib, rats were challenged intratracheally with HNE by means of nebulization for 30 days. The expression of mucin (MUC) 5AC, protein kinase C (PKC), and extracellular signal-regulated kinase 1/2 (ERK1/2) was assessed by ELISA, RT-PCR or Western blotting. The results showed that scutellarin inhibited MUC5AC mRNA and protein expressions induced by HNE in a concentration-dependent manner in vitro. In the in vivo model, scutellarin significantly attenuated MUC5AC mRNA expression and goblet cell hyperplasia in rats treated with HNE for 30 days, as well as decreased the phosporylation of PKC and ERK1/2 compared to the HNE control group. Therefore, our study showed that scutellarin could prevent mucus hypersecretion by inhibiting the PKC-ERK signaling pathway. Inhalation scutellarin may be valuable in the treatment of chronic inflammatory lung disease.

IDENTIFICATION OF A NEUTROPHIL ELASTASE CLEAVAGE SITE ON THE Act -CHAIN OF PRIMATE FIBRINOGEN

1987 ◽  
Author(s):  
J Weitz ◽  
S Landman ◽  
S Birken
Keyword(s):  
Neutrophil Elastase ◽  
Cleavage Site ◽  
High Performance ◽  
Human Neutrophil Elastase ◽  
Amino Acid Sequence Analysis ◽  
Specific Radioimmunoassay ◽  
Human Thrombin ◽  
The Mean

Human neutrophil elastase (HNE) cleaves the Aα21-22 bond of fibrinogen thus releasing the fibrinopeptide A (FPA)-containing fragment Aαl-21. Plasma Aal-21 levels reflect in vivo HNE activity and peptide levels are increased in cigarette smokers and patients with chronic lung disease. To further explore the HNE-fibrinogen interaction, we set out to develop an animal model. The digestion of purified baboon and marmoset fibrinogen by human thrombin, HNE and extracts of baboon and marmoset neutrophils was monitored with a specific radioimmunoassay for human FPA. Thrcmbin produced quantitative release (2 mol/mol fibrinogen) of FPA. In contrast, HNE and the neutrophil extracts did not release FPA, but rather, produced quantitative release of a larger, FPA-containing fragment. Immunochemically, this fragment was clearly distinguishable from FPA in that in vitro thrombin treatment increased its immunoreactivity 1,000-fold (thrombin increasable FPA or TIFPA). TIFPA release by the neutrophil extracts was blocked by α1-proteinase inhibitor, a specific HNE inhibitor (MeO-Suc-Ala2-Pro-ValCH2Cl) and an anti-HNE IgG, indicating that elastase was the responsible proteinase and that there was homology between the human and primate enzymes. The products of HNE and neutrophil extract proteolysis of the primate fibrinogens were then separated by high performance liquid chromatography and the TIFPA-containing fractions were subjected to amino acid sequence analysis. The FPA-containing fragments each consisted of 21 amino acids, had minor substitutions when compared with human A α] -21 [Baboon: Aα(3) Ser - Thr; Marmoset Aα(l) Ala - Thr, Aα(3) Ser - Thr, Aα(ll) Glu - Ala], and exhibited complete crossreactivity with the human peptide. Using the TIFPA assay, there was good recovery of primate or human Aαl-21 added to primate blood and the mean peptide level in 8 healthy marmosets was similar to that in man (0.5 nM and 0.4 nM, respectively). In conclusion, (1) the Aα;21 -22 bond of baboon and marmoset fibrinogen is a cleavage site for human and primate elastase, (2) baboon and marmoset Aal-21 can be measured with the assay for the human peptide, and (3) the primate serves as a useful model for the study of elastase-fibrinogen interactions.

scholarly journals Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2188-2195 ◽  
Author(s):  
RC Woodman ◽  
PH Reinhardt ◽  
S Kanwar ◽  
FL Johnston ◽  
P Kubes
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Neutrophil Migration ◽  
Specific Inhibitor ◽  
Neutrophil Infiltration ◽  
Cathepsin G ◽  
Neutrophil Adhesion ◽  
Human Neutrophil Elastase

Abstract The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

Further Sesquiterpene Lactones from Viguiera robusta and the Potential Anti-Inflammatory Activity of a Heliangolide: Inhibition of Human Neutrophil Elastase Release

2008 ◽  
Vol 63 (7-8) ◽  
pp. 533-538 ◽  
Author(s):  
Nilton S. Arakawa ◽  
Karin Schorr ◽  
Sérgio R. Ambrósio ◽  
Irmgard Merfort ◽  
Fernando B. Da Costa
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Sesquiterpene Lactones ◽  
Human Neutrophil Elastase ◽  
Reference Drug ◽  
Aggregation Factor ◽  
Anti Inflammatory ◽  
Isolated Compound

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based on spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83± 1.96) μm for formylated bacterial tripeptide (fMLP) stimulation and (11.84±1.62) μm for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.

scholarly journals Effects of human neutrophil elastase (HNE) on neutrophil function in vitro and in inflamed microvessels

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2188-2195
Author(s):  
RC Woodman ◽  
PH Reinhardt ◽  
S Kanwar ◽  
FL Johnston ◽  
P Kubes
Keyword(s):  
Neutrophil Elastase ◽  
Human Neutrophil ◽  
Neutrophil Migration ◽  
Specific Inhibitor ◽  
Neutrophil Infiltration ◽  
Cathepsin G ◽  
Neutrophil Adhesion ◽  
Human Neutrophil Elastase

The primary objective of this study was to test the hypothesis that human neutrophil elastase (HNE) affects neutrophil infiltration (adhesion and emigration) into inflamed vessels. To determine whether HNE contributes to neutrophil adhesion in vivo, intravital microscopy was used to study neutrophil-endothelial cell interactions in single inflamed postcapillary venules. Superfusion of platelet-activating factor (PAF) (100 nmol/L) onto the mesentery caused an increase in neutrophil-neutrophil interactions, neutrophil adhesion to postcapillary venules, and cellular emigration out of the vasculature. Both L658 758 (an elastase-specific inhibitor), and Eglin C (an elastase and cathepsin G inhibitor) significantly attenuated all of these parameters in vivo. To further characterize the mechanism(s) involved, various in vitro parameters were assessed. HNE, but not trypsin, caused a dose-dependent (0.01 to 1.0 microgram/mL) increase in the expression of the beta subunit (CD18) of the CD11/CD18 adhesive glycoprotein complex on neutrophils. An HNE-dependent increase in CD11b expression was also observed; however, HNE did not affect the expression of other neutrophil adhesion molecules (L-selectin), superoxide production, or degranulation. PAF-enhanced CD18 expression on neutrophils and neutrophil migration were both abolished by L658 758 but PAF-induced neutrophil adhesion to endothelial monolayers was not affected by the antiproteinase. The in vitro data suggest that the antiproteinases do not directly prevent neutrophil adhesion in vivo but may be important in other CD18-dependent events such as neutrophil- neutrophil interaction or neutrophil infiltration (chemotaxis). These results translate into an important, rate-limiting role for elastase in the process of leukocyte infiltration and accumulation in inflamed microvessels.

scholarly journals β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

2018 ◽  
Author(s):  
Weilong Shang ◽  
Yifan Rao ◽  
Ying Zheng ◽  
Yi Yang ◽  
Qiwen Hu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Proinflammatory Cytokine ◽  
Abscess Formation ◽  
Global Regulator ◽  
Methicillin Resistant ◽  
Cytokine Levels ◽  
Mrsa Infection ◽  
Subinhibitory Concentrations

AbstractMethicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections worldwide. MRSA resists nearly all β-lactam antibiotics that have a bactericidal activity and a signal inducer effect. However, studies have yet to clarify whether the inducer effect of empirically used β-lactams stimulates MRSA pathogenicity in vivo. Here, we showed that a new cluster of tandem lipoprotein genes (tlpps) was upregulated in MRSA in response to the subinhibitory concentrations of β-lactam induction. The increased Tlpps significantly altered immune responses by macrophages with high IL-6 and TNFα levels. The deletion of the tlpps mutant (N315Δtlpps) significantly decreased the proinflammatory cytokine levels in vitro and in vivo. The bacterial loads of N315Δtlpps in the mouse kidney were also reduced compared with those of the wild type N315. The β-lactam-treated MRSA exacerbated cutaneous infections with increased lesion size, extended illness, and flake-like abscess-formation compared with those of the nontreatment. The β-lactam antibiotics that promoted the MRSA pathogenicity were SarA dependent, and the increasing expression of tlpps after β-lactam treatment was directly controlled by the global regulator SarA. Overall, our findings suggested that β-lactams should be used carefully because it might lead to a worse outcome of MRSA infection than inaction in the treatment.Author summaryβ-lactams are widely used in practice to treat infectious diseases, however, β-lactams worsening the outcome of a certain disease is poorly understood. In this study, we have identified a new cluster of tandem lipoprotein genes (tlpps) that is upregulated in the major clinically prevalent MRSA clones in response to the subinhibitory concentrations of β-lactams induction. The major highlight in this work is that β-lactams induce SarA expression, and then SarA directly binds to the tlpp cluster promoter region and upregulates the tlpp expression in MRSA. Moreover, the β-lactam stimulated Tlpps are important virulence factors that enhance MRSA pathogenicity. The deletion of the tlpps mutant significantly decreases the proinflammatory cytokine levels in vitro and in vivo. The β-lactam induced Tlpps enhance the host inflammatory responses by triggering the expression of IL-6 and TNFα, thereby promoting bacterial colonization and abscess formation. These data elucidate that β-lactams can worsen the outcome of MRSA infection through the induction of tlpps that are controlled by the global regulator SarA.

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