In vitro production of monoclonal antibodies under serum-free conditions using a compact and inexpensive hollow fibre cell culture unit.

A compact and easily portable hollow fibre cell culture system using commercially available components is described. The construction is relatively cheap and simple. As the hollow fibre cell culture cartridge we chose an inexpensive haemodialyser. Though not specially developed for this purpose this performed excellently in our system. Using a serum-free medium supplemented with ethanolamine, selenium and transferrin, an average antibody production of 30-200 mg per cartridge per day could be achieved, depending on the cell line. Because a serum-free medium was used, monoclonal antibodies could readily be purified on a large scale.

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.

A new, sensitive assay to determine immunological adjuvant activity based on the immunogenicity of neuraminidase-treated sheep erythrocytes.

A novel, sensitive system to determine immunological adjuvant activity is presented. It is based on the direct haemagglutinin response of mice to neuraminidase-treated sheep red blood cells (asialo-SRBC) seven days after i.p. immunization. For two model adjuvants it is shown that the response is more sensitive to stimulation than that to normal SRBC. Optimal stimulatory activity was measured at an antigen dose of 3 x 10(6) asialo-SRBC. Using this dose stimulation indices up to 100 were observed. The minimal effective dose of dextran sulphate, the so far most potent adjuvant in the model, was only 1 microgram. It is further shown that, in addition to substances with a rather general immunostimulatory activity, compounds with adjuvanticity which is commonly restricted to cellular responses are also effective in the system. The latter and reduced activity of the model adjuvants in nude mice strongly suggest that adjuvanticity in the asialo-SRBC model is T cell-dependent. Suppression of adjuvant activity in cobra venom factor-pretreated animals may indicate an involvement of complement in extrinsic immunostimulatory activity. Results show that the asialo-SRBC model is very suitable for evaluation and mechanistic study of immunological adjuvant activity.

Effects of immunological adjuvants on the mouse complement system--II. Anti-complementary effects of surface-active compounds.

The anti-complementary effects of the surface-active immunological adjuvants dimethyldioctadecylammonium bromide (DDA) and pluronic polyols L101 and L121 were investigated in the mouse system. All three adjuvants showed complement (C)-inactivating effects. DDA caused a time- and dose-dependent reduction of alternative pathway (AP) and overall C activity, which varied with the serum concentration. Polyols induced a preferential inactivation of the AP by a more direct mechanism. A rather general, causative relationship between anti-complementary and immunological adjuvant activities is suggested. This might involve interference with nonspecific elimination of antigen, counteraction of immunosuppression by terminal C components, and/or moderation of C3b-mediated reduction of Ia-expression, leading to a better antigen presentation.

Simple sugars with affinity for the macrophage asialoglycoprotein receptor are adjuvants for the humoral immune response to neuraminidase-treated sheep erythrocytes.

The influence of monosaccharides on the humoral immune response of mice to normal and neuraminidase-treated sheep red blood cells (SRBC) was investigated. In these studies, both the sugars and antigen were administered i.p. D-Galactose displayed adjuvant activity for neuraminidase-treated but not for normal SRBC. This activity was optimal at an antigen dose of 3 X 10(6) cells. Other monosaccharides with an axial hydroxyl group at position 4 and D-mannose-6-phosphate behaved like D-galactose, whereas structurally unrelated sugars did not. The adjuvant activity of the saccharides corresponded to the affinity of the substances for the asialo-glycoprotein receptor. The adjuvant effects were also expressed in cobra venom factor-treated and C5-deficient mice. This suggests that blockade of the asialo-receptor rather than complement is involved in this form of immunoadjuvant activity. The findings are also in support of our hypothesis that obstruction of nonimmune antigen elimination, in this case at the level of macrophages, would be one of the mechanisms underlying immunologic adjuvant activity.

Study of the optimal reaction conditions for assay of the mouse alternative complement pathway.

The optimal reaction conditions for hemolytic assay of alternative complement pathway activity in mouse serum were investigated. A microtiter system was used, in which a number of 7.5 X 10(6) rabbit erythrocytes per test well appeared to be optimal. Rabbit erythrocytes were superior as target cells over erythrocytes from a number of other animal species. The optimal conditions were as follows: an incubation temperature of 39 degrees C, an ionic strength of about 200 mM, and a magnesium concentration of 2.5 mM. Incubation during 60 min was not sufficient for an end-point titration. Addition of 1 mg of zymosan A per test well, however, enhanced and accelerated the hemolytic activity of mouse serum via the alternative pathway resulting in a maximum value after 45 min. This, most probably, proceeded by a mechanism involving the formation of a zymosan-C5-convertase and bystander lysis of the target cells. In contrast to the normal alternative pathway assay the zymosan-potentiated test did, most probably, not involve natural antibodies. Cobra venom factor was more efficient in enhancing the sensitivity of the assay for the mouse alternative complement pathway than zymosan. This makes this factor very useful for testing C-poor body fluids.

Increased local complement levels upon intraperitoneal injection of mice with Listeria monocytogenes and regulation by polyanions.

Alternative complement pathway activity in mouse peritoneal fluid was determined by a sensitive microtiter assay with rabbit erythrocytes as target cells and cobra venom factor as the inducer of bystander hemolysis. The intra- and interstrain variations in five mouse strains were 30% and, maximally, 64%, respectively. No activity was detected in genetically C5-deficient mice. In C5-sufficient mice, intraperitoneal injection of heat-inactivated Listeria monocytogenes resulted in a 250% increase in local complement activity within 30 min. This effect was reversed by simultaneous injection of dextran sulfate but not heparin. These results are discussed in relation to the ability of listeriae to activate the mouse alternative complement pathway in vitro and the effectiveness of dextran sulfate and the failure of heparin in functioning as an adjuvant in the induction of a protective response in mice by a heat-inactivated listeria vaccine.

The selective localization of B lymphocytes in the spleen and the role of complement receptors.

The role of complement receptors on the localization of T and B cells in the spleen of mice was studied using short-term homing experiments in cobra venom factor (CoF)-treated animals. The localization ratio of B and T cells in the spleen of CoF-treated mice decreased significantly compared to control recipients. No changes could be found in the relative distribution of resident T and B cells in the spleen or other lymphoid organs of CoF-treated animals and when their spleen or lymph node cells were transferred, the localization pattern was normal. When cells were incubated in serum prior to transfer a disturbed localization ratio in the spleen of untreated recipients was observed. This was due to a blockade of complement receptors as determined by the inability of the incubated cells to form EAC rosettes. No blockade of EAC rosettes and no changes in localization ratios upon transfer could be observed when the cells were incubated in functionally C3-depleted serum. The results suggest a role for the complement-receptor on B cells in the initial localization in the spleen, whereas no influence upon the selective localization in high endothelial venules-bearing organs was found.

Anti-complementary amines are immunological adjuvants in mice.

Complement (C) inactivation by ammonia, ethylenediamine and methylamine in mouse serum was studied in relation to a possible adjuvant effect of the substances in a cell-mediated immune response. The amines caused a dose-dependent depletion of both alternative pathway (AP) and overall C activity in vitro and showed also pronounced adjuvant effects in the delayed type hypersensitivity response of mice to SRBC. A significant correlation between momentary inhibition of AP activity and adjuvanticity was observed (r = 0.9995; P approximately 0.02), suggesting a causative relationship between these two phenomena. Both effects seem to be a direct function of the number of amino-groups per molecule. Since, on the other hand, lysosomotropic activity of amines is known to decrease with the number of amino-residues, our findings exclude an important role of direct phagocyte inhibition in the immuno-adjuvanticity of these compounds. A longer persistence and improved presentation of antigen as indirect result of local C-depletion could account for the immunological adjuvant effects of amines.

Surface-associated sialic acid is an immunological adjuvant.

The influence of neuraminidase on the immunogenicity of heterologous erythrocytes as determined by serum haemagglutination titres was investigated in mice. For this study sheep and rabbit erythrocytes were selected because of their high and low N-acetylneuraminic (sialic) acid content, respectively. Preincubation with neuraminidase resulted in a ten-fold reduction of the immunogenicity of sheep erythrocytes (ShE). By contrast, the immune response to rabbit erythrocytes appeared to be resistant to sialidase treatment. Addition of the extrinsic adjuvant dimethyldioctadecylammonium bromide largely restored the immunogenicity of neuraminidase-treated ShE, but did not change the response to control-treated ShE. The maximal antibody level induced by neuraminidase-treated ShE was lower than that provoked by control ShE. These results suggest that sialic acid is both an intrinsic immunological adjuvant and an antigenic determinant of ShE. The adjuvant effect of sialic acid does not depend on complement component C3 as judged by the response of cobra venom factor-pretreated animals. In genetically C5-deficient and in nude mice, however, sialic acid showed diminished and absent adjuvant activity, respectively.

Analytic study of the differential anticomplementary effects of dextran sulphate and heparin in the assay for the mouse alternative pathway.

In a recent paper, a linkage between immunological adjuvant activity in mice and in vitro anticomplementary (alternative pathway assay) effects was described for different polyanions. This connection was found only if mouse serum was used as a complement (C) source. In order to investigate the possible role of C in adjuvant activity, the differential effects of polyanions on mouse C were studied in detail. For this study, substances with different activity were selected, namely dextran sulphate with strong C-regulatory and immunoadjuvant activities, and heparin, which was weakly anticomplementary and devoid of adjuvant effect. In general, studies of mouse C are complicated by the unavailability of isolation procedures for the C-components involved. This difficulty was circumvented by making use of C5-deficient serum and of the haemolytic activity of mouse membrane attack complexes formed in the fluid phase. With yeast cells as alternative pathway activators it was shown that the effect of heparin on this pathway was restricted to activation of the terminal route. In contrast, dextran sulphate also caused a functional decay of a yeast-bound alternative pathway C5-convertase and interfered with the haemolytic activity of fluid-phase membrane attack complexes as well. Further studies will be needed to decide whether these specific effects of dextran sulphate are related to the immunological adjuvant activity of the substance in mice.

Complement inhibitory and anticoagulant activities of fractionated heparins.

Almost monodisperse heparin fractions (Mw/Mn less than 1.1) were obtained by gel filtration of a commercial heparin. These fractions were assayed for anticoagulant activity (thrombin times and APTT), chromogenic anti-factor Xa activity, inhibitory activity for the human classical complement pathway, carboxyl group content and total sulfate content. Linear relationships were observed between the molecular weight of the heparin fractions and the anti-coagulant activities as determined by thrombin time- and APTT-assay and the classical complement pathway inhibitory activity. On the other hand a hyperbolic-like relationship was observed between the molecular weight of the heparin fractions and the chromogenic anti-factor Xa activity. The heparin fractions did not show significant differences with respect to the carboxyl group and total sulfate content. Low- and high affinity heparin fractions were obtained by affinity chromatography using immobilized AT III. High- and low-affinity fractions greatly differed not only with respect to their APTT activity, but also where their complement-inhibitory activities were concerned. The latter in contrast to literature data available. These differences could not be explained by the observed differences in molecular weight of high and low affinity heparin respectively.

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.

Effects of immunological adjuvants on the mouse complement system. I. The inability of the polyanion heparin to act as an adjuvant is paralleled by inefficient alternative complement pathway inhibition.

Interference with hemolytic complement activity by polyanionic substances was studied in relation to the ability of these compounds to act as an adjuvant for a dead listeria vaccine. Heparin appeared a poor inhibitor of the mouse alternative pathway not only in contrast to its effects on the mouse classical and the human classical and alternative pathways, but also when compared to two polyanions with known adjuvant activity: dextran sulfate and suramin. For the three polyanions mentioned a correlation between adjuvant activity and mouse alternative pathway inhibition was observed. These findings suggest a possible causal relationship between interference with alternative complement pathway activation and adjuvant activity.