Cr Jenkin

Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose

A simple and rapid method for isolating pure mouse IgG1, IgG2a and IgG2b immunoglobulins in nearly 100% yield is described. Mouse serum was fractionated on protein A-Sepharose 4B and the recovery of immunoglobulins was measured as a function of pH. When serum was applied to the column at pH 8.0, IgM, IgA and IgE were almost quantitatively recovered in the effluent together with non-immunoglobulin serum components. Providing the binding capacity of the column was not exceeded, essentially all IgG was retained at pH 8.0 and this could not be eluted by washing. Using buffers of decreasing pH, IgG1, IgG2a and IgG2b were sequentially eluted at pH 6.0–7.0, pH 4.5–5.0 and pH 3.5–4.0, respectively.

The development of resistance in different inbred strains of mice to infection with nematospiroides dubius.

Summary Infection by the intestinal nematode parasite Nematospiroides dubius was studied in seven different inbred mouse strains. Although there was some minor variation in the susceptibility of the different strains to a primary infection there were marked differences in their ability to develop resistance to infection following repeated exposure to infective larvae. The strains of mice which developed the best resistance also expelled adult worms arising from the previous infections. The adult worms resulting from a primary infection were slowly eliminated in two inbred strains studied whereas no loss occurred from outbred LACA mice. Although males and females of two strains, C3H/HeJand CBA/H were equally susceptible to a primary infection, the females developed better resistance than the male mice following two oral administrations of third stage larvae. Infected mice of every strain and both sexes contained high levels of IgG1, in the serum.

Particle recognition by haemocytes from the colonial ascidianBotrylloides leachii: Evidence that theB. leachii HA-2 agglutinin is opsonic

SummaryHaemocytes from the ascidianBotrylloides leachii were observed in vivo to phagocytose sheep erythrocytes. The possibility that a sheep erythrocyte agglutinin (the HA-2 agglutinin) previously purified fromB. leachii haemolymph functions as a recognition molecule for the phagocytosis of these erythrocytes was investigated.Untreated sheep erythrocytes were found to adhere toB. leachii haemocytes in vitro. Adherence appeared to be mediated by the HA-2 agglutinin, as evidenced by the inhibition of adhesion by lactose (which is a specific inhibitor of the HA-2 agglutinin) and by an anti-HA-2 IgG preparation. Immunofluorescence studies indicated that HA-2 molecules secreted by the haemocytes bound to unsensitised erythrocytes, causing them to adhere to haemocytes. No HA-2 agglutinin could be detected on the surface of the haemocytes in the absence of erythrocytes but receptors for the agglutinin were detected. The results suggest that the HA-2 agglutinin can function as a recognition molecule for sheep erythrocytes and other particles bearing the appropriate carbohydrate moieties on their surfaces. At least one of two other lectins purified from haemolymph (HA-1 and LBP-3) was detected by immunofluorescence on the surface of haemocytes. The function(2) of these latter molecules, neither of which binds to sheep erythrocytes, is not known.

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Abstract Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.

Molecular Basis of Self/Non-self Discrimination in the Invertebrata

In 1907 Reudiger and Davis claimed that hemolymph from several different species of invertebrates promoted the ingestion of bacteria by human polymorphonuclear leukocytes and suggested on this basis that invertebrates may possess factors (opsonins) which play an important role in enchancing phagocytosis. This particular field lay relatively dormant for almost 60 years until Tripp (1966) showed in vitro that hemolymph from the oyster Crassostrea virginica increased the rate of phagocytosis of erythrocytes by amebocytes from this animal. Two years later Stuart (1968) found that the blood cells of the octopus Eledone cirrosa would phagocytose erythrocytes only if they had been pretreated with hemolymph. Unfortunately, in neither investigation was the specificity of these factors investigated.

Ascidian haemagglutinins: Incidence in various species, binding specificities and preliminary characterisation of selected agglutinins

Abstract 1. 1. Haemolymph from 22 different ascidian species was titrated for agglutinating activity against a panel of erythrocytes from five different vertebrate species. All but three haemolymph samples possessed detectable haemagglutinins, although considerable variations in titre were recorded. The divalent cation requirements of the agglutinins were determined. 2. 2.|The binding specificity and size of the agglutinins was investigated by sugar inhibition assays and gel filtration in order to determine if any of the agglutinins resembled the HA-1 and HA-2 agglutinins characterised previously from the styelid ascidian Botrylloides leachii . 3. 3.|Haemolymph samples from three other styelid ascidians contained molecules similar in size, binding specificity and divalent cation requirement to the HA-1. One of these also contained an “HA-2 like” molecule. 4. 4.|An agglutinin similar in size but with a specificity distinct from that of the HA-1 was detected in haemolymph from Halocynthia hispida , an ascidian belonging to a different family but within the same order as B. leachii .

Identification of the HA-2 agglutinin in the haemolymph of the ascidian Botrylloidesleachii as the factor promoting adhesion of sheep erythrocytes to mouse macrophages

The HA-2 agglutinin purified from B. leachii haemolymph is shown to mediate the binding of sheep erythrocytes to mouse macrophages. This agglutinin appears to be wholly responsible for the adhesive activity of haemolymph, since upon chromatography of the latter through Sephadex G-200, the adhesive activity for both sheep and guinea pig erythrocytes was confined to those fractions containing the HA-2 agglutinin. The HA-1 agglutinin recovered in earlier fractions was unable to promote the adhesion of guinea pig erythrocytes to macrophages. Adsorption experiments indicated that this was due to a lack of ligand sites on the macrophage surface. These data support earlier conclusions that the HA-1 and HA-2 agglutinins have distinct binding specificities.

Reduced infectivity of Nematospiroides dubius larvae after incubation in vitro with neutrophils or eosinophils from infected mice and a lack of effect by neutrophils from normal mice

Summary Neutrophils and eosinophils, isolated from the blood of mice infected with Nematospiroides dubius, were tested for their capacity to damage exsheathed third stage N. dubius larvae in vitro. In the presence of fresh serum from infected mice, both types of granulocyte caused a significant reduction in larval infectivity (up to 40–50%) whereas lymphocytes/monocytes prepared from the same blood samples were inactive. Neutrophils were at least as active as eosinophils, on a cell for cell basis. None of the cells exhibited larvicidal activity in the absence of serum and serum alone had no effect. The reduction in larval infectivity caused by neutrophils in the presence of fresh normal mouse serum (NMS) was only marginally less than that obtained using immune mouse serum (IMS), suggesting that complement, which is activated by the larvae via the alternative pathway and mediates the adherence of both cell types, was able to promote the larvicidal effect of these cells in vitro. In contrast to neutrophils, eosinophils were considerably less effective in NMS than in IMS. Both NMS and IMS were ineffective if they had been heat‐inactivated or incubated with methylamine at pH 8.0 to destroy complement activity. The immunoglobulin fraction of IMS was also ineffective in promoting neutrophil or eosinophil‐mediated larval damage. These results indicate that in this in vitro system antibodies are incapable of directing the activity of either cell type in the absence of complement. A novel finding of this study was that neutrophils from uninfected mice were unable to reduce larval infectivity in the presence of fresh NMS or IMS. ‘Altered’ neutrophils possessing larvicidal activity appeared in the blood of mice within 4 days of infection with N. dubius.

ACTIVATION OF COMPLEMENT BY TETRATHYRIDIA OF MESOCESTOIDES CORTI: ENHANCEMENT BY ANTIBODIES FROM INFECTED MICE AND LACK OF EFFECT ON PARASITE VIABILITY

Tetrathyridia of the cestode Mesocestoides corti were isolated from the peritoneal cavity of infected mice. The parasites activated guinea pig and mouse complement (C) in vitro by both the classical and alternative pathways as shown by quantitative C fixation and crossed immunoelectrophoresis. The ability of tetrathyridia to activate mouse C was enhanced by preincubating the parasites in serum obtained from mice infected with M. corti. Antibodies of the IgG1 class, an immunoglobulin found in profoundly increased amounts in mice infected with M. corti, as well as IgM and IgG2 antibodies, bound to cultured tetrathyridia and facilitated deposition of the third component of C (C3) from dilute mouse serum, presumably via classical pathway activation. The results demonstrate that mouse IgG1 antibodies do not prevent the activation of C by the tetrathyridia or by C-fixing antibodies of other classes which become attached to the tetrathyridia. The activation of C in vitro by tetrathyridia did not affect their ability to grow in mice, even though C3-derived polypeptides could be detected by immunofluorescence on the surface of the parasites.