Jan M.N. Willers

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.

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.

Effects of variation in time and dose of cyclophosphamide injection on delayed hypersensitivity and antibody formation

Abstract A single injection of Cyclophosphamide (CY) in a dose of 300 mg of CY/kg of mice resulted in enhanced delayed hypersensitivity (DH) when administered between at least 7 days prior to and 15 days after intracutaneous (ic) immunization of sheep red blood cells in Freunds complete adjuvant. The maximal enhancement occurred when CY was applied 8 hr before the antigen. Using the latter interval, the effect of varying the dose of CY before ic or intraperitoneal (ip) injections of antigen was studied. Combined with ic immunization, increasing doses of CY resulted in increasing DH. The ip route of immunization needed CY in amounts of at least 100 mg/kg to augment DH to a detectable level. The enhancing effect of lower doses of CY was more pronounced when the interval between immunizing and eliciting injections was reduced. Administration of 300 or 200 mg of CY/kg before ip immunization suppressed the antibody formation, when measured S and 7 days later. A dose of 100 mg of CY/kg caused a suppression of antibody formation on Day 5, but an enhancement on Day 7. With this dose, a maximal enhancement of DH was found on both days. The results suggest that CY interferes with more than one regulatory mechanism of the immune response.

Adjuvant Effect of Nonionic Block Polymer Surfactants in Humoral and Cellular Immunity

The adjuvant activities of four chemically similar, but physicochemically different nonionic surface-active agents called pluronic polyols F 68, L 31, L 101 and L 121 were studied. These four agents were tested in mice using an experimental model developed for studying the adjuvant activity of the cationic surface-active agent dimethyl dioctadecyl ammonium bromide (DDA). L 121 and DDA enhanced the primary antibody response to sheep red blood cells (SRBC) while F 68, L 31 and L 101 suppressed this response. The secondary humoral response to SRBC was enhanced by the polyol L 121 while the secondary response to dinitrophenylated bovine serum albumin (DNP22-BSA) was enhanced by both L 121 and L 101. DDA and the polyol L 101 were very effective adjuvants for induction of delayed-type hypersensitivity (DTH) to SRBC and DNP22-BSA after intracutaneous immunization of mice with a mixture of antigen and adjuvant. Since the four pluronic polyols were composed of identical chemical constituents, we proposed that difference in their activities as adjuvants were due to variation in their physicochemical properties. A correlation was found between a physicochemical parameter, the hydrophilelipophile balance (HLB), and the adjuvant activities of the pluronic polyols and several other types of nonionic surface-active agents. The agents which were strong adjuvants all had HLB values within a narrow range which classified them as spreading agents.

Endotoxin-induced antitumor activity in the mouse is highly potentiated by muramyl dipeptide

The ability of aqueous solutions of various endotoxin preparations, muramyl dipeptide (MDP) and combinations of endotoxin and MDP, to induce necrosis and regression of subcutaneous Meth A transplants in mice and their toxicity were studied. While intravenously injected toxic endotoxins, in contrast to a detoxified preparation and to MDP, induced considerable necrosis and regression of their own, addition of MDP potentiated the antitumor potential of both toxic and detoxified endotoxins to the same high degree. Detoxified endotoxin combined with MDP, however, was far less toxic than toxic preparations alone or combined with MDP. This indicates that toxicity is not directly related to therapeutic potential.

Antitumour activity of endotoxin, concanavalin A and poly I: C and their ability to elicit tumour necrosis factor, cytostatic factors, and interferon in vivo

SummaryConcanavalin A, endotoxin, poly I : C, and tumour necrosis serum (TNS) were compared for antitumour activity against Meth A sarcoma transplanted in syngeneic BALB/c mice and their capacity to induce tumour necrosis factor (TNF), heat-stable cytostatic factors, and heat-labile interferon in the blood of normal and Corynebacterium parvum-pretreated mice. All the agents induced hyperemia and inhibition of mitosis at 4 h, and by 24 h many tumours had a dark necrotic centre. Subsequent tumour growth was inhibited and in some of the treated mice tumours regressed completely. Poly A : U and normal mouse serum did not induce regression and their effects were less marked in all other respects, suggesting that these events may be linked. The necrotizing effects of concanavalin A and poly I : C are unlikely to be mediated by TNF, because neither agent could mimic endotoxin in eliciting RNase-resistant necrotizing and regressing activity in the serum of mice pretreated with C. parvum. Poly I : C did not induce strong cytostatic activity in the sera of C. parvum-treated mice, and for this and other reasons these factors are unlikely to be responsible for the observed effects. Concanavalin A, endotoxin, and poly I : C induced high levels of serum interferon but purified interferon had only weak antitumour activity in the Meth A system, suggesting that interferon may not be the mediator.From these and other data it is concluded that there is no clear relationship between the capacity of the agents to induce tumour necrosis and their capacity to elicit TNF, cytostatic factors, and interferon.

Cellular and Humoral Adjuvant Activity of A Mistletoe Extract

The adjuvanticity of the mistletoe preparation Iscador was investigated. The cellular response to sheep red blood cells (SRBC) was augmented after intracutaneous immunization with antigen and different doses of Iscador. Iscador did not change the cellular response to 2 x 10(7) intraperitoneally administered SRBC. The IgM plaque forming cell response was accelerated and followed by an augmentation of the IgG and IgA plaque forming cell response. Evidence is presented that the immunogenic and inflammatory capacities of Iscador contribute to its adjuvant activity. Both micro-organisms and soluble, filter-adherent constituents in Iscador possess adjuvant activity. The relation between the immunostimulating properties of Iscador and its anti-tumour activity is discussed.

Killed Listeria monocytogenes vaccine becomes protective on addition of polyanions

IN the interaction between macrophages and intracellular parasites, microbial components which prevent phagosome–lysosome fusion1,2 have a decisive role. The resistance to faculative intracellular parasite Listeria monocytogenes is a form of cell-mediated immunity3, as it can be passively transferred with viable lymphoid cells4. Resistance to listeria infection can be induced by sublethal numbers of viable listeria5,6. Induction of resistance by non-viable listeria, however, is effective only after repeated injections of listeria preparations in combination with lipopolysaccharides7. Lipopolysaccharide is cytotoxic against macro-phages8,9, suggesting that the difference in processing of viable and dead microorganisms by macrophages might explain the induction of resistance by vaccine of live and not of dead bacteria. Impairing macrophage activity might result in a processing of dead listeria advantageous for the induction of resistance. We show here that killed L. monocytogenes vaccine becomes protective when the polyanions dextran sulphate (DS 500, molecular weight 500,000, Serva) and suramin (Bayer) are added. These polyanions are known to inhibit phagosome–lysosome fusion in macrophages2,10. Moreover, dextran sulphate is a potent adjuvant for both humoral11 and cell-mediated responses12. The latter might be particularly important in view of the requirement of cell-mediated immunity for resistance to listeria3.

Endotoxin-induced release of tumour necrosis factor and interferon in vivo is inhibited by prior adrenoceptor blockade

SummaryThe effect of α- and β-adrenoceptor blocking agents on endotoxin-induced release of tumour necrosis factor (TNF), and of interferon in the circulation of Corynebacterium parvum-treated mice was the subject of this study. TNF was quantified after injection of TNF containing heated serum (TNS) into Meth A sarcoma-bearing mice by determining colour, extent, and incidence of haemorrhagic necrosis. The release of TNF was weakly inhibited by the competitive α-blocker phentolamine and the β-blocker propranolol. The non-competitive α-blocker phenoxybenzamine inhibited to a higher degree. Endotoxin-induced elicitation of growth-inhibiting principles into TNS was antagonized by propranolol and phenoxybenzamine. Administration of adrenaline before endotoxin inhibited the elicitation of TNF and growth-inhibitory activities, which indicates tachyphylaxis. The release of interferon was effectively inhibited by both α-adrenoceptor blockers but not by propranolol. The interferon was heat-labile. The results indicate that endotoxin-induced TNF and interferon are separate factors, elicited in different ways. As both α-blockers do not only inhibit reactions at the α-adrenergic receptor but also reactions at the serotonin receptor and in the case of phenoxybenzamine also at the choline receptor, it is suggested that endotoxin-induced release of the anti-tumour factors is controlled by reactions mediated by one or more of these receptors. It is suggested that the inhibition of TNF release by propranolol may be due to the membrane-stabilizing activity of this agent.

Combinations of two synthetic adjuvants: Synergistic effects of a surfactant and a polyanion on the humoral immune response

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.