The activity of macrophages and lymphocytes appears to be abnormal in RZB/B1 mice. Depressed T- cell dependent activity has been noted both here and elsewhere. However, whilst Graft - versus - Host reactions are reported elsewhere to be depressed also, cell transfer experiments within the strain or to histocompatible strains lead to splenomegally in the recipients. Evidence is given which suggests that either a type of Graft - versus - Host or Host - versus - Graft reaction occurs. The abnormal behaviour of the macrophages in this strain reported here may play an important part in the autoimmunity and the abnormal immune capacity of NZB/B1 mice. Phagocytosis was observed to increase as the mice aged, but was accompanied by a decrease in the degradative ability of these P.E macrophages. Hyperactivity in phagocytosis and hypoactivity in the degradation of antigen were obvious when these activities were compared with those of non autoimmune strains of mice. The possible causes of this aberrant behaviour are appraised as is an aetiology of a genetic or viral origin.

No description available.

Ovine IgG, IgM and IgA and antisera specific for these immunoglobulins were prepared. These reagents were used to estimate immunoglobulin levels in sheep sera and certain other body fluids including the respiratory secretions. The results showed that a secretory IgA system existed in the ovine respiratory tract. Furthermore IgA antibodies to Mycoplasma ovipneumonia were identified in the lung fluid of a sheep clinically affected with pulmonary adenomatosis. IgA antibodies specific for Parainfluenza 3 were demonstrated in the respiratory secretions of lambs which had been experimentally infected with the virus. A large molecular weight non-immunoglobulin substance, which inhibited Parainfluenza 3 and three other paramyxoviruses, was also identified in the respiratory secretions of both conventionally reared and specific pathogen free lambs. It is suggested that in certain reports non-specific inhibitors present in the nasal secretions of calves may have been confused with Parainfluenza 3 specific IgA antibody. Attempts were made to define the protective role of nasal secretion antibody with vaccination-challenge experiments in specific pathogen free lambs. It was shown that live Parainfluenza 3 administered intranasally stimulated comparable serum antibody titres but higher nasal secretion titres than the same dose of live virus given intramuscularly. Inactivated virus inoculated without adjuvant by either route stimulated low or undetectable serum titres and no nasal antibody. Immunity to aerosol challenge, as assessed by viral shedding from the nose and changes in post challenge antibody titres, was best conferred by intranasal inoculation with live virus. Hence there was some evidence that the presence of antibody in the nasal secretions reduced the susceptibility to infection. In subsequent experiments it was found that intramuscular inoculation of inactivated Parainfluenza 3 in complete Freund's adjuvant stimulated high serum and nasal secretion titres, which protected against challenge. However the nasal secretion antibody was IgG^, which was possibly selectively transferred from serum. This contrasted with the earlier finding of IgA antibodies in the respiratory secretions after intranasal inoculation of live virus and showed that a second mucous antibody system existed in the ovine respiratory tract. Results from an experiment with young colostrum fed lambs indicated that maternal antibody alone could prevent infection with Parainfluenza 3 virus. This showed that the presence of cell-mediated immunity may not be essential to prevent sheep becoming infected with this virus. In this and a subsequent experiment it was demonstrated that colostral IgG passed into the nasal and lachrymal secretions of young lambs. This finding supported the earlier suggestion that IgG1 is selectively transferred from serum into the nasal secretions of young lambs. It is suggested that the presence of maternal IgG1 antibodies in the respiratory secretions of newborn suckled lambs could constitute an important defence mechanism against respiratory infections before local antibody synthesis begins at about two weeks of age.

This project was concerned with, certain immune responses mediated by T lymphocytes against major histocompatibility antigens of the rat. A cytotoxic population of lymph node calls was raised by grafting allogeneic skin and removing the draining lymph nodes eight days later. Those cells were incubated on a monolayer of allogeneic lymphocytes which had been finely attached to a polystyrene Petri dish pro-treated with poly-l-lyeino. The immune cells were physically separated into a major non-adherent fraction and a minor adherent fraction. Most of the latter could be removed for testing with an EDTA solution. In confirmation of other work, it was found that the non-adherent fraction was selectively depleted of cytotoxic activity; moreover the adherent fraction was selectively enriched for this activity. The antigenic specificity of the separation was confirmed by using monolayars of lymphocytes from different inbred strains. Unexpectedly, the small number of lymph node cells adherent to a third-party alloantigen were significantly less cytotoxic than the starting population. The main point of the work was to measure, as precisely as possible, the graft-versus-host (GvH) activity of the adherent and non-adherent cell populations in order to determine whether GvH activity was partitioned in the same way, as was cytotoxic activity. Simultaneous measurements of the two T cell activities produced decisive and were within the limits of detection, there was no partition of GvH activity, since the adherent cells, the non-adherent cells and the starting population were all precisely equal in this respect. The simplest explanation for these results requires three assumptions: (a) In an immune population GvH reactive cells and cytotoxic calls belong to separate subsets; (b) Although GvH reactive cells display surface receptors for alloantigens, these are not able to bring about partition in the in vitro system described here; (c) The cytotoxic and GvH reactive cells do not interact in the functional tests. These assumptions were generally supported, but not definitely proved, by a number of supplementary experiments including' measurement of GvH activity against 'third-party' alloantigens. The GvH activity of nonimmune lymph node cells was also identical in the adherent and nonadherent subpopulations. A radioisotopic labeling method, which has been used to estimate the proportion of T cells responding to a given major alloantigen complex in a systemic GvH reaction, was modified in order to estimate the proportion of immune cells which adhered to allogenic monolayers as a consequence of antigen recognition. A population of cells which had been immunised against one major alloantigen was labelled with either 3H- or 14C-uridine, and a population immune against a third-party alloantigen was labelled with the alternative isotope. The ratio of 3H to 14C in the adherent fraction, particularly that subfraction which was eluted with EDTA, always reflected the increased binding of the homologous immune population. On average, approximately 3% of the uridine-labelled coils were specifically adherent. This is of the same order as estimates of the proportion of cytotoxic cells in immune populations. However, when similar ?? were performed with immune populations labelled in vitro, with .7 H- or 14C thymidino, it was found that, on average, 2 of the MA nynthocising population was specifically adherent. This suggests that most of the specifically adherent cells belong to the actively proliferating population, which others have shown to include most of the cytotoxic activity at this stage of the response. Nevertheless, the prooiso relationship between specific adherence in vitro, and cytotoxicity has not boon determined. Several explanations are considered for the in vitro observation that cytotoxic T cells selectively adhere to the appropriate antigens while GvH reactive cells do not. The individual receptors for antigen may have higher affinity or, alternatively, the cytotoxic cell may have higher avidity because it has a greater density of receptors. A third explanation is that the cytotoxic cell reacts to the engagement of its receptors by a non-specific increase in its adhesiveness. The only possibility which these experiments rule out is that the cytotoxic cell is more adherent before encountering antigen and that antigen-specific binding adds to this to make the total adherence to allogenic cells greater than is the case with GvH reactive cells.

Killed vaccines of the adjuvant Corynebacterium parvum have proved to be potent anti-tumour agents in a number of experimental systems and are currently being assessed clinically, either alone or as an adjunct to other cancer treatments. In animals, systemically administered C. parvum can exert a strong, non-specific anti-tumour action which is thought to be mediated principally by activated macrophages. The author carried out in vitro experiments analysing tho possible pathways loading to the production of such activated maorophages in C. parvum-treated CBA mice. The tumour studied was a methylcholanthrene-induced fibrosarcoma. It was found that spleen cells from mice treated intravenously, or intraperitoneally (but not subcutaneously) with C. parvum could induce, in vitro, anti-tumour cytotoxicity in normal, non-cytotoxic, murino peritoneal macrophages, provided that C. parvum was also present. This requirement for the presence of C. parvum was shown to be a specific one, other strains of C.parvum or other unrelated bacteria being ineffective. The macrophage-activating cell was found to be radio-sensitive, but resistant to treatment with sodium aurothiomalate or anti-macrophage serum. However, exposure of normal peritoneal macrophages to sodium aurothiomalate, either before or during activation with spleen cells, abrogated the cytotoxic effects. Nylon wool column separation of spleen cells into B cell-rich and B cell-depleted fractions indicated a possible requirement for B cells in the activation process. On the other hand a T cell requirement was shown by the failure of spleen cells from C. parvum-treated T cell-deprived mice to activate macrophages in vitro. Treatment of spleen cells with anti-9 serum prior to the activation process yielded inconclusive evidence with regard to the T cell dependence of macrophage activation. Spleen cells capable of activating macrophages in vitro did not appear in the C. parvum-treated mouse until after day 6 post C. parvum infection. This contrasted with the early appearance, by day 3, of cytotoxic spleen and peritoneal cells in intraperitoneal treated mice. This suggested the existence in vivo of two separate pathways leading to the stimulation of anti-tumour effector cells. In vitro induction of anti-tumour activity could also be achieved using locally-stimulated lymph node cells, or cell-free supernatants from mixed cultures of C. parvum-sensitised spleen cells and C. parvum. During the course of in vitro studies on C. parvum-activated macrophages it was observed that such macrophages lost their cytotoxic capacity on culture. This loss of activity could be delayed by the addition of C. parvum to the cultures. This maintenance effect by C. parvum was found to be radio-resistant, sensitive to sodium aurothiomalate, T cell dependent, and at least partially specific. The significance of these results and the possible mechanisms underlying them have been discussed in relation to the existing knowledge of C. parvum's biological and anti-tumour effects.

In developing a vaccine against Rhipicephalus appendiculatus, an important tick ectoparasite of livestock in Africa, a necessary first step is identification of antigens which give protective immunity. Antigen profiles of extracts of unfed immature and adult Rh. appendiculatus ticks and their fractions were compared by immunoblotting with sera raised against tick infestation and against whole or fractionated extracts. Several antigens (51.5, 40, 36.5 and 23kDa) were observed to be absent in extracts of fed or partially fed adult ticks. Antigens of 84, 60 and 40kDa were consistently detected in extract fractions shown in immunisation/tick challenge experiments to be immunogenic. The 60kDa antigen was found both in soluble and membrane fractions, whilst the 84 and 40kDa antigens did not appear to be membrane associated. The 84 and 40kDa species appeared heavily glycosylated with a broad range of carbohydrate moieties being present. The 60kDa antigen did not bind significantly to any of the lectins used, suggesting only minimal glycosylation. Probing extracts of unfed larval ticks of different species with serum raised against an immunogenic fraction of Rh. appendiculatus unfed nymphal extract revealed 60kDa antigen species in each of the five different tick species. Immunostaining of sections of unfed adult female Rh. appendiculatus illustrated marked differences in the distribution of antigens associated with adult tick feed and those associated with immunisation with extracts of unfed nymphal ticks and their fractions. Of the unfed immature Rh. appendiculatus extract fractions used in immunisation and adult tick challenge feed experiments in guinea pigs, SEHPLC fraction 2 and the 45% ammonium sulphate supernate fraction were found to be the most protective. The prospects for developing an anti-Rh. appendiculatus vaccine are discussed, and antigen purification strategies are suggested.

No description available.

The object of this thesis was to develop and characterise monoclonal antibodies which would recognise damaged and or regenerating endothelial cells and which might therefore be useful in the diagnosis and treatment of vascular disease. The work described was carried out with cultured vascular endothelial cells of human, bovine, or porcine origin. In vitro models of endothelial damage were devised in which cells were damaged by various methods. Mouse monoclonal antibodies were raised by inoculation with cultured endothelial cells and tested against damaged endothelium in vitro. A number of antibody producing clones were identified, isolated and grown on for characterisation of the antibody produced. Using an immunohistochemical system for localising the sites of binding of these antibodies to cultured endothelial cells, a number of different patterns of antibody binding were identified. Attention was concentrated on antibodies which appeared to selectively bind to damaged endothelial cells. In an attempt to identify the antigens recognised by the monoclonal antibodies the protein constituents of endothelial cell lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protein bands were transferred to nitrocellulose membranes so that specific bands which reacted with individual protein bands could be identified. A number of protein bands were identified which reacted with specific monoclonals. An interaction between damaged endothelial cells and the Fc component of IgG have been previously described in the literature. The non-specific interaction of IgG with endothelial cells was confirmed in this study and the binding component identified. Reasons for believing that the monoclonal antibodies raised in this project predominantly interacted with damaged endothelial components by specific rather than non-specific binding are discussed.

No description available.

B cells internalize antigen in a specific manner through the B cell receptor (BCR). The antigen is processed into peptides that are loaded on to MHC class II molecules and presented to CD4+ T cells. I have investigated factors that affect how the antigen is taken up, processed and presented. One way that B cells can obtain antigen is by extracting antigen that is tethered on cells in the form of immune complexes. Follicular dendritic cells (FDCs) are thought to be a type of cell that can provide antigen in this way. B cell acquisition of intact antigen from FDCs is thought to be important during the maturation of an immune response. As previous studies are relative limited due to the technical difficulties of obtaining FDCs, the molecular mechanisms involved in this antigen transfer are still unclear. I have therefore attempted to develop an in vitro system for assessing antigen transfer from FDCs isolated from murine spleen to B cells, with the aim of investigating the molecular requirements for transfer of antigen from FDCs to B cells. In particular, I have studied the role of the BCR on B cells and complement receptors 1 and 2 (CR/2) on FDCs in uptake of antigen by B cells from FDCs. I have also used soluable antigen to investigate the role of affinity in uptake and presentation of antigen by B cells. I have used a panel of recombinant antigens, together with B cell transfectants expressing antigen-specific BCRs, to study BCR-antigen interactions of varying affinities. T cell hybridomas recognising different epitopes of the antigen allowed me to determine the efficiency of antigen presentation. A high affinity interaction between the BCR and its antigen generally anables more effective presentation than a low affinity one. I have found, however, that the effect of BCR-antigen affinity varies according to the T cell epitopestudied. Under certain circumstances presentatiion may be better when the affinity of the BCR for the antigen is reduced.