H. van Dijk

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.

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 activity of an aqueous extract of Azadirachta indica stem bark

The interference of an aqueous extract of the stem bark of Azadirachta indica with different parts of the human immune system was investigated. The extract showed strong anticomplementary effects which were dose-and time-dependent and most pronounced in the classical complement pathway assay. Moreover, a dose-dependent decrease in the chemiluminescence of polymorphonuclear leukocytes was observed and a dose-dependent increase in the production of migration inhibition factor by lymphocytes.

Two functionally and chemically distinct immunomodulatory compounds in the gel of aloe vera

An aqueous extract of Aloe vera gel was analyzed guided by modulatory activity with regard to the in vitro activation of human complement and of human polymorphnuclear leucocytes (PMN). Upon ultrafiltration a high (h-Mr) and a low (l-Mr) molecular mass fraction were obtained. Pre-incubation of human pooled serum with the h-Mr fraction resulted in a depletion of classical and alternative pathway complement activity. In contrast, only the l-Mr fraction could inhibit the production of free oxygen radicals by activated PMNs. The latter activity cannot be attributed to non-specific effects like toxicity, interference with stimulant binding or scavenger activity.

Estimation of classical pathway of mouse complement activity by use of sensitized rabbit erythrocytes.

A simple photometric assay was devised for determining classical complement pathway activity in mouse serum using sensitized rabbit erythrocytes as target cells. These cells appeared more sensitive to lysis by mouse complement than sensitized mouse and sheep erythrocytes, most probably by their ability to escape the C3b inactivator system. Advantages of the assay over other techniques are the high sensitivity and the avoidance of the use of radioisotopes. With this test it is possible to get more insight in the complement system of an animal species that has been most widely in use in immunological research.

Delayed and Exaggerated Postprandial Complement Component 3 Response in Familial Combined Hyperlipidemia

Very low density lipoprotein overproduction is the major metabolic characteristic in familial combined hyperlipidemia (FCHL). Peripheral handling of free fatty acids (FFAs) in vitro may be impaired in FCHL by decreased action of acylation-stimulating protein (ASP), which is identical to the immunologically inactive complement component 3a (C3adesArg). Because decreased FFA uptake by impaired complement component 3 (C3) response (as the precursor for ASP) may result in enhanced FFA flux to the liver in FCHL, we have evaluated postprandial C3 changes in vivo in FCHL patients. Accordingly, 10 untreated FCHL patients and 10 matched control subjects underwent an oral fat loading test. Fasting plasma C3 and ASP levels were higher in FCHL patients (1.33±0.09 g/L and 70.53±4.37 mmol/L, respectively) than in control subjects (0.91±0.03 g/L and 43.21±8.96 mmol/L, respectively;P =0.01 and P <0.05). In control subjects, C3 concentrations increased significantly after 4 hours (to 1.03±0.04 g/L). In FCHL, plasma C3 was unchanged after 4 hours. The earliest postprandial C3 rise in FCHL patients occurred after 8 hours (1.64±0.12 g/L). The maximal apolipoprotein B-48 concentration was reached after 6 hours in FCHL patients and control subjects. Postprandial FFA and hydroxybutyric acid (as a marker of hepatic FFA oxidation) were significantly higher in FCHL patients than in control subjects, and the early postprandial C3 rise was negatively correlated with the postprandial FFA and hydroxybutyric acid concentrations. The present data suggest an impaired postprandial plasma C3 response in FCHL patients, most likely as a result of a delayed response by C3, as the precursor for the biologically active ASP, acting on FFA metabolism. Therefore, an impaired postprandial C3 response may be associated with impaired peripheral postprandial FFA uptake and, consequently, lead to increased hepatic FFA flux and very low density lipoprotein overproduction.

Effects of low molecular constituents from Aloe vera gel on oxidative metabolism and cytotoxic and bactericidal activities of human neutrophils

In traditional South-East Asian medicine the therapeutic value of the parenchymous leaf-gel of Aloe vera for inflammatory-based diseases is well-reputed. The aim of this study is to investigate at which level gel-constituents exert their activity. We show here that low -Mr constituents of an aqueous gel-extract inhibit the release of reactive oxygen species (ROS) by PMA-stimulated human PMN. The compounds inhibit the ROS-dependent extracellular effects of PMN such as lysis of red blood cells. The capacity of the PMN to phagocytose and kill micro-organisms at the intracellular level is not affected. The inhibitory activity of the low-Mr compounds is most pronounced in the PMA-induced ROS production, but is significantly antagonized by the Ca-ionophore A23187. It is shown that the inhibitory effect of the low-Mr compounds is the indirect result of the diminished availability of intracellular free Ca-ions.

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.

Inhibitory activity of Jatropha multifida latex on classical complement pathway activity in human serum mediated by a calcium-binding proanthocyanidin

This study isolates and characterizes the anti-complement constituent(s) present in the latex of Jatropha multifida, in an attempt to explain the traditional application of the latex in the treatment of infected wounds. Guided by the inhibition of classical pathway (CP) complement activity in human serum, a polymer was isolated which could be characterized as a proanthocyanidin. The polymer inhibits CP activation of the complement cascade, while alternative pathway (AP) activation is relatively insensitive to the polymer. This is due to the selective depletion of Ca2+, but not Mg2+, from the incubation medium.

Imunodulatory compounds from Picrorhiza kurroa: Isolation and characterization of two anti-complementary polymeric fractions from an aqueous root extract

Two aqueous root extracts of Picrorhiza kurroa, one prepared by extraction at 4 degrees C and the other by refluxing, were purified using the guidance of modulation of classical (CP) and alternative (AP) pathway complement activity. By means of methanol extraction and gel filtration chromatography, two polymeric fractions were isolated from the cold water extract. A methanol-soluble polymeric fraction (CS1) was highly active in inhibiting CP complement activity exclusively, whereas a methanol-insoluble polymeric fraction (CI1) exhibited an inhibitory effect on both CP and AP complement activity. Preliminary chemical analysis of the anti-complementary fractions revealed the presence of structures of carbohydrate and of peptide nature in CS1 and CI1. The modulation of CP complement activity by CS1 was studied in more detail. Its inhibitory effect was proven to be based on complement consumption rather than on chelation of Ca2+ and/or Mg2+ or on stabilization of the target cells in the complement-assay. The purification of the aqueous extract prepared by refluxing, resulted in the isolation of a polymeric fraction with the same qualities as CS1. However, a fraction with properties similar to CI1 could not be isolated from this extract.