C. J. Beukelman

Microassay for colorimetric estimation of complement activity in guinea pig, human and mouse serum.

A sensitive colorimetric microassay for determining haemolytic complement activity was devised. It is carried out in U-welled microtitre dishes covered with plastic tape, which are incubated in a waterbath and subsequently centrifuged. The supernatant is transferred to flat-bottomed microtitre dishes and haemolysis is estimated by automatic measuring of the absorption using an interference filter of 405 nm in a Titertek Multiskan. Advantages of the method described are saving time and materials, and avoiding the use of radioactive nuclides. This microassay may therefore be a useful substitute for macro and semi-micro tests for colorimetric determination of serum complement activity and for microassays based on the release of a radio-isotope.

Apocynin inhibits peroxynitrite formation by murine macrophages

Peroxynitrite (ONOO−) the highly reactive coupling product of nitric oxide and superoxide, has been implicated in the pathogenesis of an increasing number of (inflammatory) diseases. At present, however, selective peroxynitrite antagonizing agents with therapeutic potential are not available. Therefore, the NADPH‐oxidase inhibitor apocynin (4‐hydroxy‐3‐methoxy‐acetophenone) was tested for its ability to inhibit peroxynitrite formation in vitro The murine macrophage cell‐line J774A.1, stimulated with IFNγ/LPS, was used as a model. Conversion of 123‐dihydrorhodamine (123‐DHR) to its oxidation product 123‐rhodamine was used to measure peroxynitrite production. Stimulated peroxynitrite formation could be completely inhibited by apocynin, by the superoxide scavenger TEMPO as well as by the nitric oxide synthase inhibitor aminoguanidine. Apocynin and aminoguanidine specifically inhibited superoxide and nitric oxide formation respectively as confirmed by measuring lucigenin enhanced chemiluminescence and nitrite accumulation. It is concluded that J774A.1 macrophages produce significant amounts of peroxynitrite, which is associated with nitric oxide production and NADPH‐oxidase dependent superoxide formation. The NADPH‐oxidase inhibitor apocynin proved to be a potent inhibitor of both superoxide and peroxynitrite formation by macrophages, which may be of future therapeutic significance in a wide range of inflammatory disorders.

Curcacycline A — a novel cyclic octapeptide isolated from the latex of Jatropha curcas L.

From the latex of Jatropha curcas L. (Euphorbiaceae) a novel cyclic octapeptide was isolated, which we named curcacycline A. The compound was found to contain one threonine, one valine, two glycine, and four leucine residues. By two‐dimensional 1H‐NMR spectroscopy (HOHAHA and ROESY), its sequence was determined to be Gly1‐Leu2‐Leu3‐Gly4‐Thr5‐Val6‐Leu7Leu8‐Gly1. Curcacycline A displays a moderate inhibition of (i) classical pathway activity of human complement and (ii) proliferation of human T‐cells.

Inhibition of human complement by beta-glycyrrhetinic acid.

Licorice, the root extract of Glycyrrhiza glabra L., is used as a medicine for various diseases. Anti‐inflammatory as well as anti‐allergic activities have been attributed to one of its main constituents, glycyrrhizin. These activities are mainly ascribed to the action of the aglycone, β‐glycyrrhetinic acid. β‐Glycyrrhetinic acid has a steroid‐like structure and is believed to have immunomodulatory properties. To determine whether interference with complement functions may contribute to the immunomodulatory activity of β‐glycyrrhetinic acid, its effects on the classical and alternative activation pathways of human complement were investigated. We found that β‐glycyrrhetinic acid is a potent inhibitor of the classical complement pathway (IC50=35 μm), whereas no inhibitory activity was observed towards the alternative pathway (IC50>2500 μm). The anticomplementary activity of β‐glycyrrhetinic acid was dependent on its conformation, since the α‐form was not active. It was also established that naturally occurring steroids, e.g. hydrocortisone and cortisone, did not inhibit human complement activity under similar conditions. Detailed mechanistic studies revealed that β‐glycyrrhetinic acid acts at the level of complement component C2.

Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora.

Extracts of the rhizomes of Picrorhiza scrophulariiflora Pennell (Scrophulariaceae) were investigated for their in vitro and in vivo immunomodulatory properties. Diethyl ether extracts showed potent inhibitory activity towards the classical pathway of the complement system, the respiratory burst of activated polymorphonuclear leukocytes, and mitogen-induced proliferation of T-lymphocytes. Furthermore, such extracts showed anti-inflammatory activity towards carrageenan-induced paw edema. No effects were observed in experimentally induced arthritis in mice.

Anti-inflammatory properties of a liposomal hydrogel with povidone-iodine (Repithel®) for wound healing in vitro

A liposomal hydrogel with 3% povidone-iodine (PVP-ILH, Repithel) has shown clinical benefit in settings where inflammation and/or reactive oxygen species are thought to impede wound healing (e.g., burns, chronic wounds and in smokers). This in vitro study investigated whether PVP-ILH is able to reduce inflammatory events responsible for the impairment of the wound healing process in such patients. Therefore, the following assays were conducted with PVP-ILH (and derived control hydrogels to identify the component responsible for the effect): inhibition of reactive oxygen species production by human polymorphonuclear neutrophils (PMNs) and in a cell-free system, oxygen consumption assay of PMNs (prior to oxidative burst), inhibition of human complement (limiting the generation of complement factors), mast cell degranulation, nitric oxide production by murine macrophages and TNF-alpha production by human monocytes/macrophages. Where toxicity could cause cell inhibition, cell viability was assessed. PVP-ILH and its components interacted in our series of bioassays at various stages in the inflammation cascade. Scavenging of superoxide anions was the most pronounced effect. Furthermore, povidone-iodine inhibited PMN production of reactive oxygen species (inhibition of oxygen consumption) and a mast cell inhibitory (stabilising) activity was observed. Based on these results, the clinically observed, beneficial wound healing effects of PVP-ILH may also be attributed to an impediment of inflammatory activity, mainly by iodines free radical scavenging. Controlling oxidative stress in the wound may be of great importance, especially since further reactions as, e.g., the formation of peroxynitrite from NO and ROS are prevented.

A one-step isolation procedure for phospholipase A2-free cobra venom factor by fast protein liquid chromatography

A very rapid and efficient procedure for isolation of cobra venom factor (CoF) from Naja naja venom is presented. The method is based on Mono Q anion exchange chromatography on a system for fast protein liquid chromatography (FPLC). CoF was eluted by a buffer of pH 7.4 at 280 mM salt. A purification of 33.7 X was reached with a yield of at least 27%. Contamination with phospholipase was under the detection limit of a sensitive radiometric assay (less than 25 ppm), while the starting material contained 5%. The preparation displays high C-depleting activity in vivo.

Podacycline A and B, two cyclic peptides in the latex of Jatropha podagrica

Two novel cyclic peptides were isolated from the latex of Jatropha podagrica, which we named podacycline A and B. Podacycline A is a cyclic nonapeptide with the sequence Gly1-Leu2-Leu3-Gly4-Ala5-Val6-Trp7-Ala8-Gly9+ ++-Gly1. The sequence of podacycline B, a cyclic heptapeptide, was determined to be Phe1-Ala2-Gly3-Thr4-Ile5-Phe6-Gly7-Phe1. The amino acid residues of both compounds were found to have the L-configuration.

New properties of wheat bran: anti‐biofilm activity and interference with bacteria quorum‐sensing systems

Some plant extracts, have been demonstrated to interfere with the microbial metabolism of several pathogenic bacteria. Within this antimicrobial properties it has been described the potential to inhibit or destroy biofilms or to interfere in quorum-sensing (QS) systems. However, to our knowledge, no study exploring this potential of wheat-bran (WB) has been published. The purpose of the present study is to evaluate the anti-biofilm activity of WB against a cow mastitis strain of Staphylococcus aureus and also its possible interference with bacterial QS systems. The potential of inhibition and destruction of the biofilm was studied by different in vitro assays. Also, we tested the ability of WB to interfere in bacterial QS by degrading acyl-homoserine lactones (AHL) as one of the most studied QS signal molecules for Gram-negative bacteria. The soluble extract of WB at 0.5% showed anti-biofilm activity, inhibiting biofilm formation and also destroying it. Similarly, the > 300 kDa fraction from WB had significant anti-biofilm activity in both in vitro assays. The WB also showed a potential to interfere with bacterial QS systems, as it was demonstrated to contain certain lactonase activity able to reduce AHL concentration in the medium. The present study reveals two additional beneficial properties of WB extract never explored before, which may be related to the presence of defence compounds in the plant extract able to interfere with microbial biofilms and also QS systems.

In vitro immunomodulatory activity of Filipendula ulmaria

Extracts of the roots, herb and flowers of Filipendula ulmaria (L.) Maxim. were investigated for in vitro immunomodulatory properties. Strong inhibitory activity was found towards the classical pathway of complement in the ethyl acetate extracts of roots and flowers, in all methanol extracts and in the aqueous root extract. Except for the light‐petroleum extracts, all fractions tested inhibited the production of reactive oxygen species by human polymorphonuclear leukocytes. The diethyl ether root extract was found to be most potent in inhibiting lymphocyte proliferation. The role of tannins and other constituents in these processes are discussed.