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Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system - termed PmrA/PmrB - that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments.

Sphingolipids (SLs) play essential roles in most eukaryotes, but in the trypanosomatid protozoan Leishmania major their functions differ significantly. Previously we showed that null mutants defective in de novo sphingoid base synthesis (spt2-) lacked SLs but grew well and retained lipid rafts while replicating as promastigotes in vitro. However, they experienced catastrophic defects in membrane trafficking on entry into stationary phase, and failed to differentiate to the infective metacyclic form. Here we showed this mutant retained the ability to enter macrophages silently and inhibit activation, although as expected most parasites were destroyed. However, in mouse infections, after a delay rapidly progressive lesions appeared, and purified amastigotes were fully virulent to macrophages and mice. Mass spectrometry of spt2- amastigote lipids revealed the presence of high levels of parasite-specific inositol phosphorylceramides (IPCs) not synthesized by the mammalian hosts. Inhibitor studies showed that salvage occurs at the level of complex SLs, suggesting that parasites carry out 'headgroup' remodelling. Additionally, we describe a new defect of the spt2- promastigotes involving 'empty' acidocalcisomes (ACs), which may point to the origin of this organelle from the lysosome-related organelle/multivesicular body biogenesis pathway. However, ACs in spt2- amastigotes appeared quantitatively and morphologically normal. Thus salvage of SLs and other molecules by intracellular amastigotes play key roles in AC biogenesis and parasite survival in the host.

In Salmonella enterica, the PhoP-PhoQ two-component system governs resistance to structurally different antimicrobial peptides including the alpha-helical magainin 2, the beta-sheet defensins and the cyclic lipopeptide polymyxin B. To identify the PhoP-regulated determinants mediating peptide resistance, we prepared a plasmid library from a phoP mutant, introduced it into a phoP mutant and selected for magainin-resistant clones. One of the clones harboured the PhoP-activated ugtL gene, deletion of which rendered Salmonella susceptible to magainin 2 and polymyxin B, but not defensin HNP-1. We established that ugtL encodes an inner membrane protein that promotes the formation of monophosphorylated lipid A in the lipopolysaccharide. Inactivation of both ugtL and the regulatory gene pmrA, which controls lipid A modifications required for resistance to polymxyin B (but not to magainin 2) and is post-transcriptionally activated by the PhoP-PhoQ system, resulted in a strain that was as susceptible to polymyxin B as a phoP mutant. The most frequently recovered clone harboured the yqjA gene, which we show is PhoP regulated and required for resistance to magainin 2 but not to polymyxin B or defensin HNP-1. Our results indicate that different PhoP-mediated modifications in lipid A are necessary for resistance to different antimicrobial peptides.

Intracellular mycobacteria release cell wall glycolipids into the endosomal network of infected macrophages. Here, we characterize the glycolipids of Mycobacterium bovis BCG (BCG) that are released into murine bone marrow-derived macrophages (BMMØ). Intracellularly released mycobacterial lipids were harvested from BMMØ that had been infected with 14C-labelled BCG. Released BCG lipids were resolved by thin-layer chromatography, and they migrated similarly to phosphatidylinositol dimannosides (PIM2), mono- and diphosphatidylglycerol, phosphatidylethanolamine, trehalose mono- and dimycolates and the phenolic glycolipid, mycoside B. Culture-derived BCG lipids that co-migrated with the intracellularly released lipids were purified and identified by electrospray ionization mass spectrometry. When delivered on polystyrene microspheres, fluorescently tagged BCG lipids were also released into the BMMØ, in a manner similar to release from viable or heat-killed BCG bacilli. To determine whether the released lipids elicited macrophage responses, BCG lipid-coated microspheres were delivered to interferon gamma-primed macrophages (BMMØ or thioglycollate-elicited peritoneal macrophages), and reactive nitrogen intermediates as well as tumour necrosis factor-alpha and monocyte chemoattractant protein-1 production were induced. When fractionated BCG lipids were delivered on the microspheres, PIM2 species reproduced the macrophage-activating activity of total BCG lipids. These results demonstrate that intracellular mycobacteria release a heterogeneous mix of lipids, some of which elicit the production of proinflammatory cytokines from macrophages that could potentially contribute to the granulomatous response in tuberculous diseases.

Nerve damage, resembling that caused by Mycobacterium leprae in man, was created by the injection of cobalt-irradiated M. leprae organisms into the tibial nerve of guinea-pigs. Assessment of nerve damage was made by clinical, electrophysiological and morphometric means at intervals up to 13 weeks after injection. Quantitative immunohistochemical analysis of neuropeptide-containing fibres in the skin of the foot was also carried out. Significant nerve damage occurred 3 weeks after injection of M. leprae organisms. Motor and sensory functional loss peaked at 5 weeks after injection, and there was a significant decrease of peptide-immunoreactive nerves in all skin compartments. The nerve damage was self-limiting and functional recovery had occurred by 13 weeks. The model shows many of the features found in the nerve damage of treated leprosy patients.

Biozzi (H-2dq1) AB/H mice are marked not only by their high titre antibody responses following immunization with protein antigens but are also susceptible to experimental allergic encephalomyelitis (EAE) induction. The T-cell receptor (TcR) repertoire in this recently characterized strain was analysed. Biozzi AB/H mice were found to express the Thy-1a, Ly-1b, Ly-2b and Ly-5b alleles. Serological typing of the TcR-V beta + peripheral T cells suggested that the AB/H mice belong to the TcR-V beta b haplotype and express a deletion ligand for TcR-V beta 6+ and TcR-V beta 8.1+ T cells. This was confirmed by Southern blot analysis which revealed the presence of Mtv-17, Mtv-23, Mtv-31 (Y chromosome) and notably Mtv-7. Therefore the AB/H mice are Mls-1a. Despite the depletion/absence of the majority of TcR-V beta families in EAE-resistant (BALB/c) x EAE-susceptible (AB/H) F1 mice it was possible to induce EAE in these F1 animals. This suggests either that the deleted TcR-V beta-bearing T cells were not the principal encephalitogenic cells or that the TcR-V beta usage is sufficiently heterogeneous to accommodate such deletion events, in spinal cord-induced EAE.

The expression of adhesion molecules on central nervous system (CNS) endothelia was examined during chronic relapsing experimental allergic encephalomyelitis (CREAE) in the Biozzi AB/H mouse. Active disease episodes (acute and relapse) were associated with the up-regulation of MALA-2, the murine homologue of intercellular adhesion molecule-1 (ICAM-1), on CNS endothelia and the infiltration of ICAM-1-positive mononuclear cells. In addition, the high endothelial venule (HEV)-associated MECA-325 antigen was evident in perivascular lesions, particularly in relapsing disease. The peripheral lymph node HEV-associated vascular addressin defined by MECA-79 antibody was not detectable in the CNS during CREAE. However, the mucosal HEV addressin was evident in lesions, which ultrastructurally was found to be expressed on the surface of endothelial cells by immunoelectron microscopy. The expression of adhesion molecules, such as ICAM-1, may provide a means by which both the initial neuroantigen-specific and the subsequent antigen-non specific cells extravasate into the CNS. Such infiltration may induce the expression of the vascular addressins which may then provide a means of site-selective cellular recruitment leading to disease progression.

Lymphokine (LK)-induced aggregation of peritoneal exudate cells (PEC) has been measured using a quantitative technique. Aggregating activity could be removed from LK preparations by absorption of these with PEC and, in addition, the absorbing PEC, on further incubation themselves aggregated. Absorption of aggregating activity to PEC was rapid, being easily measurable at between 2-5 and 10 min although it was difficult to demonstrate at 30 min. Trypsinized PEC were as effective as normal PEC in absorbing aggregating activity. alpha-L-fucose inhibited LK-induced PEC aggregation and this inhibition showed some specificity in being significantly greater than that obtained with D(+)galactose. However, measurement of the subsequent aggregation of PEC which had been pulse exposed to LK, in the presence of alpha-L-fucose, showed that alpha-L-fucose had no effect on this aggregation. It is concluded that the measurement of aggregating activity can be used to study LK binding to PEC, although the relationship of this binding to the inhibition of aggregation obtained with alpha-L-fucose is not clear.

Desensitization of guinea-pigs with ovalbumin (OA) or bovine gamma globulin (BGG) induces strong specific desensitization and is associated with a non-specific energy to tuberculin--PPD. Similarly, it was found that animals receiving desensitizing injections of PPD have suppressed delayed hypersensitivity reactions to OA or BGG. PPD also induced strong specific desensitization. Cyclophosphamide (CY) given in one large dose (300 mg/kg) 3 days before immunization failed to affect the specific desensitization induced by all three antigens. However, if CY was given 1 day after immunization, it was not possible to induce specific desensitization. The induction of non-specific desensitization was prevented in all three antigen systems if CY was given either 3 days before or 1 day after immunization. Desensitization with either OA or BGG markedly suppressed the specific 4 hr Arthus reactions.

Partial unresponsiveness to 2,4-dinitrofluorobenzene (DNFB) was produced by the epicutaneous application of 2 per cent 2,4-dinitrothiocyanatebenzene (DNTB) to the dorsum of the ear, 14 and 7 days before sensitization. This state of partial unresponsiveness could be broken by treatment with 300 mg/kg cyclophosphamide (CY) 3 days before the sensitization dose of DNFB. There was a significant increase in the uptake of 125I-labelled iododeoxyuridine in the draining lymph node, 6 days after the application of 2 per cent DNTB and in the contralateral nodes at 4 and 6 days. The state of partial unresponsiveness to DNFB was not associated with a decrease in T-cell proliferation in the draining lymph nodes. The generation of suppressor cells, capable of reacting in the periphery, was demonstrated by passive cell transfer into recipients sensitized to DNFB and depleted of normally produced suppressor cells by pretreatment with CY. These cells were found in spleen and peritoneal exudates of animals treated by the epicutaneous application of 2 per cent DNTB 14 and 7 days previously. It is suggested that the suppressor cells induced by contact with DNTB act by competition with effector cells at the periphery.

A comparison was made in mice between resistance to growth of a syngeneic methylcholanthrene-induced fibrosarcoma (Meth A) and specific delayed-type hypersensitivity to this tumour. Resistance could be induced in 90% of mice following a single injection of 10(4) Meth A cells. This resistance could be transferred by spleen cells but not serum. Delayed skin reactivity to ultrasonicated Meth A cells was found in both tumour-resistant and tumour-bearing mice and could be passively transferred by spleen cells and serum. These findings suggest a dissociation between tumour resistance and specific delayed-type hypersensitivity. It would appear that, in this system, delayed skin reactivity to Meth A tumour cells is mediated by humoral antibody despite a mononuclear cell infiltrate at 24 and 48 h and that this could be the reason for its lack of correlation with host resistance to tumour growth which is cell-mediated and not transferable by serum.

Previous studies have shown that endothelial cells in the central nervous system (CNS) of normal guinea-pigs constitutively express certain MHC class II determinants, whilst the expression of other determinants is apparent during the acute phase of chronic relapsing experimental allergic encephalomyelitis (CREAE). The expression of MHC class II determinants is retained by endothelial cells derived from normal guinea-pig brain tissue and maintained in culture. This present study demonstrates that the MHC class II molecules on these cells can be recognized by allogeneic lymphocytes, resulting in a proliferative response which is enhanced by the addition of exogenous IL-2. The endothelial cells were incapable of presenting either purified protein derivative or ovalbumin, but they could present autologous myelin basic protein (MBP), an encephalitogen implicated in the pathogenesis of EAE. The resulting lymphocyte proliferative response was of the same magnitude as that obtained when a control population of macrophages was used to present MBP. These results, therefore, suggest that cerebrovascular endothelia have the potential to play a role in the pathogenesis of EAE.

Haptenated murine Langerhans' cells (LCs) have been reported to induce contact sensitivity when injected via the subcutaneous, intraperitoneal and, in some instances, the intravenous route. Similar studies were undertaken to elucidate the role of the LC in the induction of contact sensitivity in the guinea-pig. The subcutaneous injection of dinitrophenylated epidermal cells induced hapten-specific contact sensitivity in a dose-dependent fashion. This contrasts with the tolerance that was induced by the intravenous or intraperitoneal injection of similarly haptenated cells. Contact sensitivity by haptenated epidermal cells could be induced in syngeneic and allogeneic recipients and did not require the transfer of viable cells. Using the monoclonal antibody MSgp2, which detects LCs, LC-enriched and LC-depleted populations were prepared by an 'indirect antibody' panning technique. It was found that a haptenated LC-depleted epidermal cell population (0.1% LC) induced the same degree of contact sensitivity or tolerance, depending on the route of immunization, as a haptenated 'freshly isolated' epidermal cell population (1% LC). Whereas, a purified population of haptenated LC (85-90%) induced no significant degree of contact sensitivity or tolerance. These results confirm our previous conclusions based the in vivo depletions of Langerhans' cells, and suggest that the epidermal Langerhans' cell is not essential for the induction of contact sensitivity in the guinea-pig. However, this does not exclude the possibility that the LC is involved in the elicitation of contact sensitivity in a sensitized animal.

The kinetics of the footpad response of bacillus Calmette-Guérin (BCG)-infected mice to soluble BCG antigens were compared in two strains of mice with different degrees of susceptibility to Mycobacterium lepraemurium. For the first 21 days the responses of the 'high-resistance' C57BL and the 'low-resistance' BALB/c to the nonpathogenic BCG were similar to that produced when the pathogenic mycobacterium was used. After 4 weeks the kinetics of the BALB/c mice changed and resembled that of the C57BL mice. The change in kinetics was compared with circulating antimycobacterial antibody levels and the response of draining lymph node cells in the antigen-specific lymphocyte transformation test. A dissociation was found between the kinetics of the delayed footpad response and the lymphocyte transformation response. Since both strains of mice are equally resistant to BCG, it appears that the delayed footpad response cannot be used as an indicator of host resistance in all mycobacterial infections.

Cyclophosphamide (300 mg/kg) given before or up to 2 days after sensitization, induces increased contact skin reactions at 8 days. Reactions were suppressed with cyclophosphamide (CY) given between 3 and 5 days after sensitization; reactivity returned on day 10. CY, given on days 6 to 8, only suppressed reactions when skin tests were made 4 days later. This temporary depression of contact sensitivity corresponds with the maximal reduction of peripheral blood lymphocytes. CY given 1-2 days after DNFB produced decreased T-cell proliferation in local lymph nodes 4 days after sensitization. CY given 3 days after DNFB produced maximal T-cell suppression on 5 day nodes. Massive increase in T-cell proliferation in 5 day nodes occurred when CY was given on the day of sensitization or the day before. Thus CY given around sensitization acts mainly on suppressor cells whereas given later, the action is principally on the effector functions.

A new quantitative method for the measurement of macrophage aggregation, induced by lymphokine preparations, is described. This involves the continuous measurement of the light absorbance of stirred macrophage suspensions. The results have been compared with those obtained by measuring macrophage migration inhibition activity for the detection of lymphokine activity. Separation of crude lymphocyte culture supernatants by Sephadex G-200 shows that the material with aggregating activity is heterogeneous. The greater amount of this activity is not separated from macrophage migration inhibition activity, and has a molecular weight of 35,000--70,000. A comparison has been made with other published methods for measuring macrophage aggregation.

Following infection subcutaneously in the footpad with 10(7) Mycobacterium lepraemurium organisms C57BL mice were able to limit multiplication of organisms at the infection site for the 6 months studied and to limit organism spread to the draining lymph node. Large numbers of organisms were present in the footpad and draining lymph node of BALB/c mice at 6 months. In spite of this difference in local immunity the changes in cellular reactivity to specific antigen as assessed by the delayed footpad response and the in vitro proliferative response of draining lymph node cells were similar in the two strains over the time studied.

The inflammatory infiltrate of the central nervous system in acute experimental allergic encephalomyelitis (EAE) in the guinea-pig has been isolated and characterized. Inflammatory cells found over the surface of the brain and spinal cord and within the leptomeninges have been isolated by washing, and were subsequently identified by a number of methods. By light microscopy approximately 60% were lymphocytes, 30% were cells of the mononuclear phagocyte series, and 5% were neutrophils: the occasional plasma cell and eosinophil were also identified. E, EA and EAC rosetting tests were used to determine the proportions of T lymphocytes (both early and late), and cells with Fc and C3 receptors respectively. Particular attention was paid to cells of the mononuclear phagocyte series bearing Fc and C3 receptors to determine whether any change or loss of these receptors was apparent. The percentage of phagocytic cells from the brain and spinal cord washes was also determined. Transmission and scanning electron microscopy showed that a large proportion of the inflammatory infiltrate consisted of cells of the mononuclear phagocyte series. This study therefore confirms that the cells of the mononuclear phagocyte series play a predominant role in EAE.

Antigen specific T lymphocyte proliferation and Lyt phenotypes of the T lymphocytes were studied in BALB/c and C57BL/6 mice infected with 10(9) M. lepraemurium organisms intravenously. A highly disseminated form of the disease developed to which all mice succumbed by 17 weeks. Maximal antigen-specific T lymphocyte proliferation was detected at 4 weeks after the infection and persisted thereafter even when the mice started to die of the infection. Accessory cells of phagocytic and adherent type did not appear to be a requirement for this proliferation. The T lymphocytes generated during the course of the infection were mostly of the Lyt 1 phenotype. However, there appeared to be no correlation between sensitized Lyt 1 cells capable of antigen-induced T lymphocyte proliferation and protective immunity.

Inflammatory cells from the meninges of guinea-pigs with acute EAE have been isolated and quantitated. A mean of 7.5 x 10(6) cells were recovered from the brains of animals showing clinical signs of disease in contrast to only 1.5 x 10(6) cells in the controls. The immunological specificity of the meningeal inflammatory cells was determined using the lymphocyte transformation test and compared with another inflammatory cell population, the peritoneal exudate cells (PECs), in the same animal. The meningeal exudate cells responded only to the mitogen Concanavalin A (Con A) and not to the antigens myelin basic protein and tuberculin (PPD). In contrast, the PECs proliferated in the presence of both antigens and the mitogen.

Effector mechanisms that produce delayed hypersensitivity reactions to tuberculin are subject to positive immunoregulation. Two different immunoregulatory mechanisms can be demonstrated. One is specific and the other, antigenic competition, is non-specific; both are sensitive to cyclophosphamide (CY). Delayed hypersensitivity to purified protein derivative PPD in guinea-pigs can be enhanced by the administration of cyclophosphamide 3 days before but not after immunization. The enhanced response seems to result from the reduced influence on effector cells of CY-sensitive suppressor cells. Passive transfer of delayed hypersensitivity to PPD is facilitated by the use of cells from CY treated animals. The response to both immunization and skin testing with ovalbumin in animals immunized with this antigen in Freud's complete adjuvant (FCA) produces a marked, non-specific reduction in the delayed hypersensitivity response to PPD. CY given 3 days before or 1 day after immunization prevents this suppression of the PPD response by antigenic competition. The data suggests that in the generation of both the specific suppressor cells for tuberculin and the non-specific suppressor cells of antigenic competition, that can influence effector cells for tuberculin, a period of rapid cell proliferation occurs that renders both mechanisms sensitive to cyclophosphamide.

A comparison has been made of the effects of cyclophosphamide (CY) pretreatment and i.v. injection of a high dose of antigen on delayed hypersensitivity induced by proteins in Freund's incomplete and complete adjuvants. Five antigens have been studied: obalbumin (OA), bovine serum albumin (BSA) bovine gammaglobulin (BGG), DNP 5-BGG and DNP 50-BGG. A spectrum of reactivity has been detected depending upon the ability of the antigen to stimulate B-cell as well as T-cell activity. Bovine serum albumin and BGG behave as relatively weak antigens in which the T-cell response, measured by delayed hypersensityity, is easily suppressed by i.v. antigen and the B-cell modulating system, revealed by CY sensitivity, is poorly stimulated. These proteins, injected i.v., are weak stimulators of antibody-dependent Arthus reactions. On the other hand, OA, DNP 5-BGG and DNP 50-BGG behave as strong antigens, resisting suppression of the T-cell response by soluble antigen and exhibiting (in Freund's incomplete adjuvant) a strongly developed CY sensitive, B-cell modulating system. Strong Arthus reactivity is readily demonstrated following i.v. administration of these antigens. A dissociation has been demonstrated between B-cell modulation of T-cell function and unresponsiveness induced by i.v. antigen. The failure to reverse the latter type of unresponsiveness by cyclophosphamide pretreatment suggests that separate mechanisms are involved in these systems.

Suppressor cells were demonstrated in the spleen of guinea-pigs made specifically unresponsive to dinitrofluorobenzene (DNFB) with dinitrobenzene sulphonic acid (DNBSO3). Transfusion of these cells at the same time as sensitization with DNFB, produced a significant reduction in the immunoblasts proliferating in the draining lymph node 4 days later. Transfusion on the day of skin testing produced no greater suppression of skin reactivity than cells taken from animals made hypo-reactive to DNFB by contact with dinitrothiocyanate benzene (DNTB). It is concluded that there are at least two sites that suppressor cells can act. In the case of total unresponsiveness induced by DNBSO3, action is both central and in the periphery. In the case of hyporeactivity induced by DNTB, in which there is no defect in proliferation of T cells in response to antigen, the action of these cells is confined to the periphery. results of spleen weight studies suggest that suppressor cells homing in the spleen respond by proliferation to epicutaneously applied DNFB.

Groups of male and female guinea-pigs were immunized with homologous sperm derived from testis (TS) or epididyimis (ES) in Freund's complete adjuvant (FCA). In vivo investigations included skin tests at 2 weeks and development of aspermatogenesis (testis weight) at 4 weeks; in vitro assays were inhibition of migration of peritoneal exudate cells (PEC) and culture of blood leucocytes (lymphocyte transformation) at weekly intervals after immunization. Antigens used were heat-treated extracts of sperm used for immunization (BTS, BES); cells were also cultured simultaneously with PPD. Skin tests revealed anergy in males as compared with females: a larger quantity of antigen which caused partial unresponsiveness in females, caused profound unresponsiveness in males although the aspermatogenesis was less severe. In vitro tests also showed anergy during the active stages of the orchitis. This was non-specific for PEC (specific unresponsiveness was not excluded), but blood leucocytes showed only specific unresponsiveness (to BES). These and previous studies suggest that the unresponsiveness results from a desensitisation by sperm antigens released during the development of aspermatogenesis.

T lymphocyte proliferation, Lyt phenotypes and their role in the evolution of protective immunity were studied in BALB/c and C57BL/6 mice infected subcutaneously with Mycobacterium lepraemurium. Antigen-induced proliferation was not demonstrable with T-enriched cells obtained from the spleens. However, these cells were capable of spontaneously proliferating in the absence of added antigen for a limited period. This proliferation was dependent on the presence of a phagocytic and adherent accessory cell. During the period when the T cells proliferated spontaneously they consisted of a mixture of Lyt-1 and Lyt-23 and were able to transfer protection to syngeneic recipient mice. Furthermore, the multiplication of the organisms was curbed during the same period demonstrating a strong association between the ability to proliferate spontaneously Lyt-1/Lyt-23 cells and protective immunity. T cells from normal BALB/c mice showed a marked suppressive effect on protection suggesting that these cells may be responsible for the susceptibility of this strain to a moderate subcutaneous infection.

Tolerance to DNCB induced by feeding guinea-pigs with the same hapten has been reversed by a single treatment with cyclophosphamide. This is paralleled by a return of the ability of T cells to proliferate in the draining lymph node following sensitization. Certain quantitative differences between this system and that previously described using intravenous DNBSO3 have been revealed. These may indicate some difference between these two mechanisms of tolerance induction.

The nature of the suppressive activity in the peritoneal exudate cells (PEC) of guinea-pigs immunized with dinitrophenyl bovine gamma globulin (DNP50-BGG) was investigated. A method was developed to isolate from the peritoneal exudate large numbers of macrophages. Using density gradient centrifugation on Percoll it was possible to obtain a population of cells which contained over 90% macrophages. This macrophage preparation was found to respond to lymphokine but to be incapable of passively transferring delayed hypersensitivity reactions. When these immune macrophages were transferred into antigen immunized animals, which had been pretreated with cyclophosphamide (CY), the skin reactions were suppressed to the same extent as when the total PEC was transferred. PEC from guinea-pigs immunized with ovalbumin in Freund's incomplete adjuvant did not suppress the skin reactions in CY-pretreated DNP50BGG immunized animals. However, in contrast, macrophages from these animals did suppress the skin reactions in the recipient guinea-pigs indicating that the macrophage suppression was not antigen specific.

Guinea-pigs were found to be fully protected from the clinical signs of EAE when they were given spinal cord in FIA either 3, 7. or 14 days immunization with spinal cord in FCA. If the animals were protected by the recruitment of suppressor cells then cyclophosphamide (CY) might break this protection. CY at 300 mg/kg and 20 mg/kg was given either before the protective inoculum to try to eliminate suppressor cell precursors or before immunization to eliminate the suppressors cells themselves. No clinical signs of EAE developed in any of these groups of guinea-pigs. It was found, however, that large numbers of meningeal inflammatory cells could be isolated from the brains of guinea-pigs protected from EAE when the protective inoculum was given only 1 week before immunization. These cell numbers were further significantly increased by giving CY in addition to the protective inoculum. The meningeal inflammatory cells and peritoneal exudate cells (PECs) from guinea-pigs protected against EAE were compared to those isolated from animals with acute EAE. These cells from guinea-pigs with acute EAE responded, in the lymphocyte transformation test, to the mitogen. The PECs also responded to the brain antigen, myelin basic protein (MBP) and to tuberculin (PPD). The meningeal inflammatory cells did not respond to either antigen. The PECs from animals protected against EAE responded to mitogen and PPD, but not to MBP, but the meningeal inflammatory cells did not respond to antigen or mitogen. The percentage of T lymphocytes in the meningeal inflammatory infiltrates of protected and unprotected guinea-pigs was similar, but that of phagocytic cells was significantly depressed in the inflammatory infiltrates of animals protected against EAE.

Cyclophosphamide (CY), which can enhance some forms of delayed-type hypersensitivity if given 3 days before immunization, is also a potent suppressor of most antibody mediated 4-h skin reactions to protein antigens. However many haemagglutinating antibodies, which are present in serum at the time of skin testing, are not similarly suppressed. Antibody titres in some sera recovered from CY-pretreated guinea-pigs differ little from titres in control sera. This resistance to CY suggests that long-lived precursors characterize the B-cell lines that produce many haemagglutinating antibodies, whereas the CY-sensitive precursors of skin reactive antibodies, which mediate Arthus-type reactions, are probably rapidly dividing, short-lived cells. Furthermore, the novel appearance of BGG antibodies in sera from CY-pretreated animals immunized with DNP50-BGG indicates that haemagglutinating antibody responses to some antigens are regulated by CY-sensitive mechanisms.

Normal thymus lymphocytes and T cells of mice have the ability to bind heat-aggregated IgG of human origin (aggHIgG), as shown by indirect immunofluorescence. At 4 degrees, the cells bind aggHIgG with an irregular speckled appearance; at 37 degrees, the aggregates are incorporated into the surface membrane, inducing rearrangement of the receptors and capping. At the point of maximum binding capacity, thymocytes show a fairly homogeneous fluorescence pattern, whereas T cells show a heterogeneous appearance. Aggregated pure human Fc fragments, but not Fab fragments, retained not only the binding capacity of the complete molecule but also the ability to induce cap formation on the surface of thymusceds, at 37 degrees.

The activation of complement by a number of metal compounds, previously found to induce chronic inflammation, was investigated. Results obtained were compared with complement activation by inulin and zymosan. It was shown that complement activation by these metal compounds did not necessarily involve either the classical or the alternative pathways. Some of these compounds were unable to activate complement in the absence of detectable plasminogen. The results obtained indicate a relationship between the ability of these compounds to induce chronic inflammation in the guinea-pig and to activate complement.

Epicutaneous exposure of mice to the contact sensitizing chemicals 4-ethoxymethylene-2-phenyl-oxazol-5-one (oxazolone) and 2,4,6-trinitrochlorobenzene (picryl chloride) causes an inhibition of proliferative responses induced following subsequent topical challenge. The effects on lymphocyte proliferation comprise both transient antigen non-specific and more persistent hapten-specific mechanisms. Pretreatment of mice with one chemical 5 days prior to sensitization with a second, at which time antigen non-specific influences on proliferative responses are manifest, results in depression of contact sensitization as measured by changes in ear thickness following challenge. If, however, the period between pretreatment and sensitization is extended the inhibition of contact sensitization disappears in parallel with a decline in the antigen non-specific depression of lymph node cell proliferation. These data reveal that there exist two homeostatic mechanisms which control proliferation in response to challenge with at least some antigens, and that the extent of lymphocyte proliferation directly influences the degree of contact sensitization achieved. Moreover these results demonstrate that, in some instances at least, competition between antigens may be a function of immunoregulatory influences on lymphocyte proliferation.

Antibodies produced in rabbits against mouse brain are able to react with the majority of lymph node and spleen murine lymphocytes, as demonstrated by indirect immunofluorescence. Cells stained by a suitably absorbed rabbit anti-mouse brain serum showed various degrees of fluorescence, from bright fluorescent rings to a few speckles per cell; lymphocytes from the spleen of deprived mice (B mice) predominantly stained in small caps. Double labelling experiments in which B cells were identified by their 'easily detectable' surface immunoglobulin revealed that a great proportion of these cells had two overlapping caps, a large one formed by the anti-immunoglobulin serum and a small one formed by the anti-brain serum. Up to 10 per cent of these spleen B cells, however, appeared as strongly fluorescent as T cells, with the suitably absorbed heterologous anti-brain serum. No anti-immunoglobulin activity was detected in this serum. It is therefore suggested that some B cells may bear on their surfaces a theta or theta-related antigen similar to that on T cells.

Inflammatory cells from the meninges of guinea-pigs with chronic relapsing experimental allergic encephalomyelitis (CREAE) have been isolated and compared to a similar cell population obtained from guinea-pigs with acute EAE. A mean of 6.2 x 10(6) cells were recovered from the brains of animals with CREAE while a similar number of cells (8.0 x 10(6)) was obtained from animals with acute EAE. Only 5.3% of the cells from animals with CREAE were found to be phagocytic in contrast with 28% of the cells obtained from animals with acute EAE. The meningeal inflammatory cells from guinea-pigs with CREAE did not respond, in the lymphocyte transformation test, to specific antigens or to mitogen. Peritoneal exudate cells (PECs) from these animals did, however, proliferate in the presence of tuberculin and of mitogen, but not with the brain antigen myelin basic protein (MBP). In contrast, the meningeal inflammatory cells from animals with acute EAE did respond to mitogen and PECs from the same animals responded to both the specific antigens and to the mitogen. The meningeal inflammatory cells and PECs from the guinea-pigs with CREAE did, however, behave in a similar manner to similar cell populations obtained from guinea-pigs fully protected against clinical signs of acute EAE.

Macrophage aggregation (measured by a light-scattering technique) induced by unpurified lymphokine preparations has been investigated. This has been shown to be Ca2+ or Mg2+ dependent and to be inhibited by a number of agents known to affect cellular physiology and biochemistry. The divalent cation ionophore A23187 causes a macrophage aggregation superficially similar to that induced by lymphokines, although differences were found. It is suggested that a biochemical response by the macrophage is required for aggregation induced by lymphokines, and thus measurements of macrophage aggregation allow a study of the early events in the response of macrophages to lymphokines.