scholarly journals In Vivo Effects of A Pro-PO System Inhibitor on the Phagocytosis of Xenorhabdus Nematophila in Galleria Mellonella Larvae

Insects ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 263 ◽  
Author(s):  
Andrea De Lerma Barbaro ◽  
Marzia B. Gariboldi ◽  
Maristella Mastore ◽  
Maurizio F. Brivio ◽  
Stefano Giovannardi
Keyword(s):  
Cell Wall ◽  
Galleria Mellonella ◽  
Mammalian Species ◽  
Specific Inhibitor ◽  
Fluorescein Isothiocyanate ◽  
Structural Features ◽  
In Vitro Tests ◽  
Xenorhabdus Nematophila

Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host’s immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host’s hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of pro-phenoloxidase (PO) system phenylthiourea (PTU), even if these effects were not observed in in vitro tests. Furthermore, biochemical modifications of X. nematophila cell wall implement in vivo phagocytosis, suggesting that this bacterium avoid phagocytosis because the ligand of phagocytic receptors is somehow buried or disguised in the cell wall. Finally, dead X. nematophila escapes engulfment even by human phagocytes suggesting that X. nematophila could be a useful model to investigate escape from phagocytosis by mammalian macrophages.

Bacterial formyl peptides affect the innate cellular antimicrobial responses of larval Galleria mellonella (Insecta: Lepidoptera)

2004 ◽  
Vol 50 (4) ◽  
pp. 279-289 ◽  
Author(s):  
Thiery B.C Alavo ◽  
Gary B Dunphy
Keyword(s):  
Bacillus Subtilis ◽  
Galleria Mellonella ◽  
Granular Cells ◽  
Xenorhabdus Nematophila ◽  
Amide Derivative ◽  
Formyl Peptide ◽  
Bacterial Stimulation ◽  
Formyl Peptides

The non-self cellular (hemocytic) responses of Galleria mellonella larvae, including the attachment to slides and the removal of the bacteria Xenorhabdus nematophila and Bacillus subtilis from the hemolymph, were affected by N-formyl peptides. Both N-formyl methionyl-leucyl-phenylalanine (fMLF) and the ester derivative decreased hemocyte adhesion in vitro, and both elevated hemocyte counts and suppressed the removal of both X. nematophila and B. subtilis from the hemolymph in vivo. The amide derivative and the antagonist tertiary-butoxy-carbonyl-methionyl-leucyl-phenylalanine (tBOC) increased hemocyte attachment to glass. The fMLF suppressed protein discharge from monolayers of granular cells with and without bacterial stimulation, while tBOC stimulated protein discharge. The peptide tBOC offset the effects of fMLF in vitro and in vivo. This is the first report implying the existence of formyl peptide receptors on insect hemocytes in which the compounds fMLF and tBOC inhibited and activated hemocyte activity, respectively.Key words: formyl peptides, hemocytes, Xenorhabdus, Bacillus.

scholarly journals Examination of Xenorhabdus nematophila Lipases in Pathogenic and Mutualistic Host Interactions Reveals a Role for xlpA in Nematode Progeny Production

2009 ◽  
Vol 76 (1) ◽  
pp. 221-229 ◽  
Author(s):  
Gregory R. Richards ◽  
Heidi Goodrich-Blair
Keyword(s):  
Manduca Sexta ◽  
Lipase Activity ◽  
Galleria Mellonella ◽  
Future Research ◽  
Broad Host Range ◽  
Xenorhabdus Nematophila ◽  
Gene Encoding ◽  
Host Interactions

ABSTRACT Xenorhabdus nematophila is a gammaproteobacterium and broad-host-range insect pathogen. It is also a symbiont of Steinernema carpocapsae, the nematode vector that transports the bacterium between insect hosts. X. nematophila produces several secreted enzymes, including hemolysins, lipases, and proteases, which are thought to contribute to virulence or nutrient acquisition for the bacterium and its nematode host in vivo. X. nematophila has two lipase activities with distinct in vitro specificities for Tween and lecithin. The gene encoding the Tween-specific lipase, xlpA, has been identified and is not required for X. nematophila virulence in one insect host, the tobacco hornworm Manduca sexta. However, the gene encoding the lecithin-specific lipase activity is not currently known. Here, we identify X. nematophila estA, a gene encoding a putative lecithinase, and show that an estA mutant lacks in vitro lipase activity against lecithin but has wild-type virulence in Manduca sexta. X. nematophila secondary-form phenotypic variants have higher in vitro lecithinase activity and estA transcript levels than do primary-form variants, and estA transcription is negatively regulated by NilR, a repressor of nematode colonization factors. We establish a role for xlpA, but not estA, in supporting production of nematode progeny during growth in Galleria mellonella insects. Future research is aimed at characterizing the biological roles of estA and xlpA in other insect hosts.

scholarly journals Lipid II-Based Antimicrobial Activity of the Lantibiotic Plantaricin C

2006 ◽  
Vol 72 (4) ◽  
pp. 2809-2814 ◽  
Author(s):  
Imke Wiedemann ◽  
Tim Böttiger ◽  
Raquel Regina Bonelli ◽  
Tanja Schneider ◽  
Hans-Georg Sahl ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Cell Wall ◽  
Pore Formation ◽  
Specific Interaction ◽  
Structural Features ◽  
Binding Motifs ◽  
Whole Cells ◽  
Lipid Ii

ABSTRACT We analyzed the mode of action of the lantibiotic plantaricin C (PlnC), produced by Lactobacillus plantarum LL441. Compared to the well-characterized type A lantibiotic nisin and type B lantibiotic mersacidin, which are both able to interact with the cell wall precursor lipid II, PlnC displays structural features of both prototypes. In this regard, we found that lipid II plays a key role in the antimicrobial activity of PlnC besides that of pore formation. The pore forming activity of PlnC in whole cells was prevented by shielding lipid II on the cell surface. However, in contrast to nisin, PlnC was not able to permeabilize Lactococcus lactis cells or to form pores in 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes supplemented with 0.1 mol% purified lipid II. This emphasized the different requirements of these lantibiotics for pore formation. Using cell wall synthesis assays, we identified PlnC as a potent inhibitor of (i) lipid II synthesis and (ii) the FemX reaction, i.e., the addition of the first Gly to the pentapeptide side chain of lipid II. As revealed by thin-layer chromatography, both reactions were clearly blocked by the formation of a PlnC-lipid I and/or PlnC-lipid II complex. On the basis of the in vivo and in vitro activities of PlnC shown in this study and the structural lipid II binding motifs described for other lantibiotics, the specific interaction of PlnC with lipid II is discussed.

scholarly journals Sulfation of fucoidin in focus embryos: III. Required for localization in the rhizoid wall

1978 ◽  
Vol 78 (3) ◽  
pp. 866-873 ◽  
Author(s):  
WE Hogsett ◽  
RS Quatrano
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Alginic Acid ◽  
Fluorescein Isothiocyanate ◽  
Sulfated Polysaccharide ◽  
Specific Region ◽  
Fucus Distichus ◽  
Rhizoid Formation

Zygotes of the brown alga Fucus distichus L. Powell accumulate a sulfated polysaccharide (fucoidin) in the cell wall at the site of rhizoid formation. Previous work indicated that zygotes grown in seawater minus sulfate do not sulfate the preformed fucan (an unsulfated fucoidin) but form rhizoids. Under these conditions, we determined whether sulfation of the fucan is required for its localization in the rhizoid wall. This was accomplished by developing a specific stain for both the fucan and fucoidin. Using a precipitin assay, we demonstrated in vitro that the lectin ricin (RCA(I)) specifically complexes with both the sulfated and desulfated polysaccharide. No precipitate is observed when either is incubated in 0.1 M D-galactose or when RCA(I) is mixed with laminarin or alginic acid, the other major polysaccharides in Fucus. RCA(I) conjugated with fluorescein isothiocyanate (FITC) is also shown to bind specifically to fucoidin using a filter paper (DE81) assay. When added to zygotes, RCA(I)-FITC binds only to the site of fucoidin localization, i.e., the rhizoid cell wall. However, RCA(I)-FITC is not observed in the rhizoid wall of zygotes grown in the absence of sulfate. This observation is not due to inability of RCA(I)-FITC to bind to the fucan in vivo. Chemically desulfated cell walls that contained fucoidin in the rhizoid wall bind RCA(I)-FITC only in the rhizoid region. Also, the concentration of fucose-containing polymers and polysaccharides that form precipitates with RCA(I) is the same in embryos grown in the presence or absence of sulfate. If sulfate is added back to cultures of zygotes grown without sulfate, fucoidin is detected at the rhizoid tip by RCA(I)-FITC several hours later. These results support the conclusion that the enzymatic sulfation of the fucan is a modification of the polysaccharide required for its localization and/or assembly into a specific region of the cell wall.

Isolation and characterization of CD39-like phosphodiesterase (Cc-PDE) from Cerastes cerastes venom: Molecular inhibitory mechanism of antiaggregation and anticoagulation.

2020 ◽  
Vol 27 ◽  
Author(s):  
Hamida Kiheli ◽  
Fatah Chérifi ◽  
Meriem Ameziani ◽  
Samah Saoud ◽  
Ghania Hariti ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Therapeutic Potential ◽  
Specific Inhibitor ◽  
Structural Features ◽  
Size Exclusion ◽  
Antiplatelet Aggregation ◽  
Isolation And Characterization ◽  
Significant Prolongation

Background: Cerastes cerastes venom contains several bioactive proteins with inhibitory potential of platelet aggregation and blood coagulation. Material and Methods: The purification process consists of three successive chromatographies including G75-Sephadex size exclusion, DEAE exchange chromatography and affinity using Sildenafil as a main PDEs’ specific inhibitor. The amino acid sequence of purified Cc-PDE was determined by liquid chromatography coupled off line to MALDI-TOF/TOF. Modeling and structural features were obtained using several bioinformatics tools. In vivo and in vitro antiplatelet aggregation and anticoagulant assays were performed. Results: Cc-PDE (73 506.42 Da) is a 654-residue single polypeptide with 1-22 signal peptide and it ischaracterized by the presence of predominant basic amino acids suitable to alkaline pI (8.17). Cc-PDE structure is composed of β-strands (17%) and α-helices (24%) and it shares a high identity with homologous snake venom PDEs. Cc-PDE hydrolyzes both Bis-pnitrophenyl phosphate (Km = 2.60 ± 0.95 mM, Vmax = 0.017 ± 0.002569 µmolmin-1 ) and p-nitrophenyl phosphate (Km = 7.13 mM ± 0.04490 mM, Vmax = 0.053 ±0.012 µmolmin-1 ). Cc-PDE prevents ADP- and ATP-induced platelet aggregation by hydrolyzing ADP and ATP, reducing surface P-selectin expression and attenuating platelet function. In addition, Cc-PDE inhibits coagulation factors involved in the intrinsic pathway demonstrated by a significant prolongation of activated partial thromboplastin time and in vivo long-lasting anticoagulation. Conclusion: The obtained results revealed that Cc-PDE may have a therapeutic potential and could be a remedy for thromboembolic diseases as an alternative of anticoagulant and antiplatelet aggregation chemical origins.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Inhibition of Fibrinolysis and Fibrinogenolysis in Man: Comparison of ε-Aminocaproic Acid and Kallikrein Inhibitor

1963 ◽  
Vol 10 (01) ◽  
pp. 106-119 ◽  
Author(s):  
E Beck ◽  
R Schmutzler ◽  
F Duckert ◽  
Keyword(s):  
Aminocaproic Acid ◽  
Side Reactions ◽  
In Vitro Tests ◽  
Factor V ◽  
Clinical Use ◽  
Strong Inhibitor ◽  
Kallikrein Inhibitor ◽  
Therapeutic Possibilities

SummaryInhibitor of kallikrein and trypsin (KI) extracted from bovine parotis was compared with ε-aminocaproic acid (EACA): both substances inhibit fibrinolysis induced with streptokinase. EACA is a strong inhibitor of fibrinolysis in concentrations higher than 0, 1 mg per ml plasma. The same amount and higher concentrations are not able to inhibit completely the proteolytic-side reactions of fibrinolysis (fibrinogenolysis, diminution of factor V, rise of fibrin-polymerization-inhibitors). KI inhibits well proteolysis of plasma components in concentrations higher than 2,5 units per ml plasma. Much higher amounts of KI are needed to inhibit fibrinolysis as demonstrated by our in vivo and in vitro tests.Combination of the two substances for clinical use is suggested. Therapeutic possibilities are discussed.

Role of in vivo and in vitro Tests in the Diagnosis of Severe Cutaneous Adverse Reactions (SCAR) to Drug

2019 ◽  
Vol 25 (36) ◽  
pp. 3872-3880 ◽  
Author(s):  
Marcel M. Bergmann ◽  
Jean-Christoph Caubet
Keyword(s):  
Adverse Reactions ◽  
Lymphocyte Transformation ◽  
Stevens Johnson Syndrome ◽  
In Vitro Tests ◽  
High Morbidity ◽  
Patch Tests ◽  
Severe Cutaneous Adverse Reactions ◽  
Cutaneous Adverse Reactions

Severe cutaneous adverse reactions (SCAR) are life-threatening conditions including acute generalized exanthematous pustulosis (AGEP), Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). Diagnosis of causative underlying drug hypersensitivity (DH) is mandatory due to the high morbidity and mortality upon re-exposure with the incriminated drug. If an underlying DH is suspected, in vivo test, including patch tests (PTs), delayed-reading intradermal tests (IDTs) and in vitro tests can be performed in selected patients for which the suspected culprit drug is mandatory, or in order to find a safe alternative treatment. Positivity of in vivo and in vitro tests in SCAR to drug varies depending on the type of reaction and the incriminated drugs. Due to the severe nature of these reactions, drug provocation test (DPT) is highly contraindicated in patients who experienced SCAR. Thus, sensitivity is based on positive test results in patients with a suggestive clinical history. Patch tests still remain the first-line diagnostic tests in the majority of patients with SCAR, followed, in case of negative results, by delayed-reading IDTs, with the exception of patients with bullous diseases where IDTs are still contra-indicated. In vitro tests have shown promising results in the diagnosis of SCAR to drug. Positivity is particularly high when the lymphocyte transformation test (LTT) is combined with cytokines and cytotoxic markers measurement (cyto-LTT), but this still has to be confirmed with larger studies. Due to the rarity of SCAR, large multi-center collaborative studies are needed to better study the sensitivity and specificity of in vivo and in vitro tests.

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