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Man has moved rapidly from the hunter-gatherer environment to the living conditions of the rich industrialized countries. The hygiene hypothesis suggests that the resulting changed and reduced pattern of exposure to microorganisms has led to disordered regulation of the immune system, and hence to increases in certain inflammatory disorders. The concept began with the allergic disorders, but there are now good reasons for extending it to autoimmunity, inflammatory bowel disease, neuroinflammatory disorders, atherosclerosis, depression associated with raised inflammatory cytokines, and some cancers. This review discusses these possibilities in the context of Darwinian medicine, which uses knowledge of evolution to cast light on human diseases. The Darwinian approach enables one to correctly identify some of the organisms that are important for the 'Hygiene' or 'Old Friends' hypothesis, and to point to the potential exploitation of these organisms or their components in novel types of prophylaxis with applications in several branches of medicine.

Background: The hygiene hypothesis is often proposed to explain the high prevalence of atopy in the western world. Dysregulation of the immune system may result from inadequate exposure to micro-organisms such as mycobacteria. A small trial suggested that a killed extract of Mycobacterium vaccae ameliorates atopic dermatitis (AD)., Objectives: To confirm in a large clinical trial whether killed M. vaccae ameliorates AD in 5-16-year-old children., Methods: This was a randomized, placebo-controlled, double-blind, multi-centre study of the effect of intradermal injection of killed M. vaccae (0.1 or 1 mg) on patients, aged 5-16, with moderate-to-severe AD. Patients were followed up for 24 weeks. The primary end point was the change in severity of AD at 12 weeks, assessed using the six area, six-sign, atopic dermatitis (SASSAD) score. Secondary end points included changes in disease extent, patient's global assessment and children's dermatology life quality index., Results: There were 166 patients randomized. The mean SASSAD score fell to a similar degree at week 12 in all treatment arms: from 33 to 24, (26%) in the high-dose group, from 30 to 23 (25%) in the low-dose group and from 36 to 27 (24%) in the placebo group (P>0.05). Secondary end points followed the same trend. Adverse events were generally those expected to occur in this population. Injection site reactions occurred in 32 patients at week 4., Conclusions: M. vaccae was no more effective than the placebo in ameliorating the severity of AD.

Background: Coexistence with harmless microorganisms such as lactobacilli, saprophytic mycobacteria and some helminths, throughout evolution, may have shaped the host immune system. Exposure to such organisms may have therapeutic benefits by triggering immunoregulatory mechanisms that control inappropriate immune responses to self, gut contents or allergens., Objective: We determined whether treatment with Mycobacterium vaccae by gavage influences the host immune response both locally and systemically. We also investigated whether delivery by this route prevents severe symptoms of disease in a murine model of pulmonary allergic inflammation., Results: A single intragastric administration of M. vaccae induced a transient increase in the production of IL-10 and IFN-gamma by mesenteric lymph nodes cells and splenocytes. In addition, in a mouse model of pulmonary allergic inflammation, a single treatment with M. vaccae by gavage not only diminished the total cellular infiltrate and the eosinophilic component induced by subsequent intratracheal allergen challenge, but also biased local and systemic cytokine production towards IL-10. Delivery of M. vaccae by gavage was as effective as subcutaneous treatment., Conclusion: This is the first report to suggest that heat-killed mycobacteria can down-regulate symptoms of allergic inflammation by the intragastric route. These data suggest an alternative route of treatment with M. vaccae for patients with allergic conditions.

Expression of interleukin (IL)-4 is increased in tuberculosis and thought to be detrimental. We show here that in healthy contacts there is increased expression of its naturally occurring antagonist, IL-4delta2 (IL-4delta2). We identified contacts by showing that their peripheral blood mononuclear cells (PBMC) released interferon (IFN)-gamma in response to the Mycobacterium tuberculosis-specific antigen 6 kDa early secretory antigenic target (ESAT-6). Fresh unstimulated PBMC from these contacts contained higher levels of mRNA encoding IL-4delta2 (P=0.002) than did cells from ESAT-6 negative donors (noncontacts). These data indicate that contact with M. tuberculosis induces unusual, previously unrecognized, immunological events. We tentatively hypothesize that progression to active disease might depend upon the underlying ratio of IL-4 to IL-4delta2.

Problems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current 6-month chemotherapy regimens used to treat tuberculosis, or of supplementing ineffective therapy. In this study we sought to define the mechanism of action of two immunotherapies, both of which have previously been shown to prolong survival. Secondly, we wished to identify any clinically useful synergy between these therapies. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by type 1 cytokines plus tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), followed by a phase of progressive disease. This progressive phase is accompanied by increasing expression of IL-4, and diminished expression of IL-1 and TNF-alpha. Animals in this late progressive phase of the disease (day 60) were treated with two injections (day 60 and day 90) of 0.1 or 1.0 mg of heat-killed Mycobacterium vaccae, or with 3beta, 17beta-androstenediol (AED; 25 microg subcutaneously three times/week), or with both therapies. We show here using four techniques in parallel (morphometry, immunohistochemistry with automated cell counting, semiquantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assays of cytokines in lung extracts) that treatment with M. vaccae causes a switch back towards a type 1 cytokine profile, restoration of expression of IL-1alpha and TNF-alpha, and a switch from pneumonia to granuloma. This is very similar to the changes previously seen after treatment with AED. However, there was no evidence for synergy between M. vaccae and AED.

The concentration of cortisol in a tissue is regulated by a reversible enzyme 'shuttle' that can deactivate cortisol by converting it to cortisone, or activate cortisone by converting it to cortisol. The activity of this shuttle, and the direction in which it operates, is regulated by numerous factors including cytokines. This results in large swings in the effective cortisol concentration in sites of inflammation at different phases of an inflammatory response. Thus changes in local cortisol concentration can be largely independent of circulating cortisol levels. The relevant shuttle enzymes are present in skin, blood vessels and nervous tissue, and inhibition of the enzymes in skin enhances the local anti-inflammatory effect of cortisol. We therefore suggest that changes in the activity or direction of action of the shuttle in leprosy lesions may predispose to reactions, requiring exogenous steroid supplements to regain control of the inflammation.

Immunity to Mycobacterium tuberculosis requires a T helper 1 (Th1) cytokine balance accompanied by tumour necrosis factor-alpha (TNF-alpha), and activated macrophages. These facets of the immune response are sensitive to suppression by glucocorticoids (GC), which can reactivate and exacerbate tuberculosis in man and animals. Dehydroepiandrosterone (DHEA) and its derivative, 3beta,17beta androstenediol (AED), are reported to have antiglucocorticoid properties in vivo. We therefore investigated the effects of predetermined optimal doses of these compounds, on the course of pulmonary tuberculosis in an established model in BALB/c mice in which an early phase of Th1-mediated response accompanied by adrenal hyperplasia, is followed by a switch to Th2, progressive loss of TNF-alpha expression and disease progression. Both compounds were protective, particularly AED which caused a fall in bacterial counts and prolonged survival. These effects correlated with the appearance within 3 days of cellular infiltrates rich in cells expressing interleukin-2 (IL-2), IL-1alpha and TNF-alpha, and with partial suppression of the switch to IL-4 producing cells that occurred in controls. AED also caused enhanced development of granulomas at 14 days, and persistence of granuloma formation to 120 days, with a corresponding suppression of areas affected by pneumonia. Much of the therapeutic effect of AED and DHEA was obtained by treating for only the first 3 weeks, which is the phase of adrenal hyperplasia. These results suggest that the ratio of GC to anti-GC steroids may play a role in the pathogenesis of tuberculosis, and further investigation could lead to novel treatment strategies.

Changes in Mycobacterium leprae-induced lymphoproliferative responses and mediator release by leprosy patients' lymphocytes were followed during multiple drug therapy (MDT). At the time of diagnosis, multibacillary (MB) patients who did not develop reactions responded to both sonicated M. leprae and synthetic disaccharide coupled to bovine serum albumin (ND-BSA) antigens, but those who would later develop reactions did not respond, even in the presence of added cytokines. The paucibacillary (PB) group initially had high responses to sonicated M. leprae but no response to ND-BSA, even in the presence of added cytokines. In the first year of treatment, the supernatants of PB patients' cell cultures contained factors that enhanced the phytohaemagglutinin (PHA) response of normal cells. In contrast, those MB patients who did not develop reactions at a later stage produced culture supernatants that were inhibitory. Interestingly, the MB patients who later developed reactions during treatment, and did not initially respond to M. leprae, produced supernatants containing enhancing factors, like those of the PB group. Later on in the treatment, all patients had the same patterns: when response to M. leprae decreased from its highest level, inhibitory factors were produced. Further studies revealed that the supernatants which inhibited the PHA response of normal cells contained the active form of transforming growth factor-beta 1, (TGF-beta 1), whatever the disease type or treatment status of the donor. These TGF-beta 1 levels correlated directly with the degree of inhibition. Similarly supernatants that neither inhibited nor enhanced PHA responses contained the highest levels of interleukin-10 (IL-10), while those from treated patients that enhanced contained the lowest levels of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma). These cytokine correlations transcended the conventional disease classification, and imply that all patients pass through a sequence of patterns of immune response during treatment. These treatment-induced changes may explain occasional reports of response patterns at variance with the 'immunological spectrum' of leprosy.

Allergic disorders are mediated by T lymphocytes secreting T helper 2 (Th2) cytokines, interleukin-4 (IL-4) and interleukin-5 (IL-5), resulting in high levels of serum immunoglobulin E (IgE) and recruitment of eosinophils. One of the treatment strategies is to downregulate the Th2 component by inducing a T helper 1 (Th1) response to the relevant allergen, because Th1 and Th2 cytokines are thought to be mutually antagonistic. In this study, we examined the effects of Mycobacterium vaccae, a potent inducer of Th1 immunity, on allergic responses in a murine model. A single injection of M. vaccae into ovalbumin (OVA)-preimmunized BALB/c mice suppressed serum IgE over a wide dose range (10(7), 10(8) or 10(9) M. vaccae). Further experiments, using 10(7) M. vaccae injected twice, showed that this treatment inhibited not only serum IgE, but also the potential for ovalbumin-induced IL-5 production by spleen cells. This non-specific ability of a mycobacterium to decrease Th2 activity, even when not presented together with the allergen, is in agreement with recent epidemiological studies on the impact of bacillus Calmette-Guérin (BCG) vaccination, and of other potent Th1 stimuli, on the incidence of atopy. The suppression of serum IgE and allergen-specific IL-5 synthesis by M. vaccae suggest that this organism is likely to have clinical application in the immunotherapy of allergy.

The role of tumour necrosis factor-alpha (TNF-alpha) in tuberculosis is paradoxical because although there is much evidence for a protective role, there is also evidence that it plays a part in the tissue damage that characterizes human disease. We have shown previously that TNF-alpha frequently induces necrosis when injected into sites undergoing delayed-type hypersensitivity (DTH) responses to mycobacterial antigen. This is dependent on CD4+ T cells. However the presence of this sensitivity to TNF-alpha-induced necrosis depended on the immunization protocol. We have tested the hypothesis that sensitivity to TNF-alpha depends on the cytokine profile of the induced T-cell response. All subcutaneous doses of mycobacterial immunogen used (10(7) to 10(9) organisms) primed spleen cells so that they secreted interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) when cultured in vitro with soluble antigen. However priming for production of IL-4 was dose dependent as in other systems, and was produced at all times from 7 to 30 days after immunization with 10(9) organisms. Time-course studies over 30 days showed that sensitivity to TNF-alpha was found in DTH sites of animals primed for IL-4 and IFN-gamma production, but not in animals primed only for the Th1 cytokines. We suggest therefore that the paradoxical role of TNF-alpha can be resolved. In 'pure' Th1 responses it may act as an additional macrophage-activating factor. In mixed Th1 + Th2 or Th0 responses it may cause tissue damage. This mixed pattern is characteristic of tuberculosis, and of the late stage of many chronic infections where elimination of the infecting organism is failing, and chronic tissue damage is seen.

An increased percentage of circulating IgG molecules that lack galactose from the oligosaccharides on the CH2 domain correlates with disease severity in tuberculosis, rheumatoid arthritis and Crohn's disease. We have recently observed that a single injection of 10(9) autoclaved Mycobacterium vaccae given to tuberculosis patients 7 days after the initiation of chemotherapy causes accelerated clinical improvement, and clearance of bacilli from the sputum. We now show that this immunotherapy also causes rapid loss of agalactosyl IgG, detectable within 14-21 days, whereas chemotherapy alone causes agalactosyl IgG to rise further for up to 2 months. There is simultaneous inhibition of the antibody response to lipoarabinomannan, and transient enhancement of the tuberculin skin-test response. These findings are compatible with a shift from antibody production towards increased cell-mediated immunity. The ideal treatment for tuberculosis would supplement a truncated course of chemotherapy with an immunotherapeutic preparation able to down-regulate the Koch phenomenon and replace it with an efficiently bactericidal mechanism. We tentatively postulate that a fall in per cent agalactosyl IgG [%Gal(0)] in tuberculosis patients may be a marker of such a change.

Tamm-Horsfall protein (THP) is the major glycoprotein component of urine, yet its biological role remains obscure. Recent reports have suggested that a concanavalin A (Con A)-binding fraction of THP from pregnancy urine can bind the cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). In order to investigate this claim in relation to THP from normal adult urine we raised monoclonal antibodies to THP and sought THP/TNF-alpha interactions in three separate assay systems. We found no evidence that THP binds to TNF-alpha under physiological conditions, but we observed that it exerts a weak, probably not physiologically relevant, but reproducible inhibitory effect on the toxicity of TNF-alpha for monolayers of L929 cells, even when the cells are pretreated with the THP, and washed before addition of the cytokine. Since our preparations of THP do not interact directly with TNF-alpha we postulated an interaction with the cells themselves, or with their extracellular matrix. The THP was found by ELISA, immunoblotting and immunohistology, to bind to as yet unidentified components of the extracellular matrix in a manner dependent on cations, pH and carbohydrates. These data, considered in the light of the published amino acid sequence and biochemical properties, suggest that THP is a member of a structural glycoprotein family known to modulate cell adhesion.

It has previously been shown that the inherently tumour necrosis factor-alpha (TNF-alpha)-sensitive L929 murine fibroblast cell line becomes much more sensitive to the cytotoxic effect of this cytokine after exposure to Mycobacterium tuberculosis in culture. In this study it is now shown that normal human cells of types likely to be involved in tuberculous lesions are affected in a similar way. Growth of normal human fibroblasts is usually stimulated by TNF-alpha in vitro, but after exposure to M. tuberculosis or to extracts of this organism, these cells are killed rather than stimulated by subsequent exposure to TNF-alpha. Similarly, human endothelial cells become susceptible to doses to TNF-alpha which do not normally affect viability. Moreover this enhancement of sensitivity to TNF-alpha is not confined to its toxicity. Endothelial cells and HeLa cells exposed to M. tuberculosis express increased levels of ICAM-1 after subsequent exposure to TNF-alpha, implying synergy between the two stimuli. It is suggested that these effects contribute to the ability of M. tuberculosis to distort the normal protective role of TNF-alpha so that the cytokine becomes detrimental to the host.

Endotoxin (LPS)-triggered release of tumour necrosis factor (TNF) from human monocytes is high for the first 3 days in culture, and then falls to a trough between Days 4 and 6. This trough is less deep if the cells are cultured in the presence of indomethacin. If monocytes are cultured in the presence of either recombinant gamma-interferon or 1,25-(OH)2 vitamin D3, their capacity for LPS-triggered TNF release is increased. Live virulent Mycobacterium tuberculosis can substitute for the LPS, and is markedly more effective as a trigger than several strains of BCG prepared in an identical manner. Since secretion of IFN-gamma and conversion of circulating 25-(OH) vitamin D3 to the active dihydroxy metabolite by interferon-activated macrophages probably occur in tuberculosis, we suggest that triggering of TNF release from the resulting activated macrophage populations might explain much of the weight loss and tissue damage that characterize this disease. IFN-gamma does not activate effective anti-mycobacterial mechanisms in human monocytes, so its role in tuberculosis may be immunopathological rather than protective.

Soluble extracts of ultrasonically disrupted mycobacteria (including Mycobacterium leprae) cause strong T-cell-dependent, Cyclosporin A-sensitive, polyclonal B-cell activation, in human peripheral blood mononuclear cell preparations. It is not enhanced by cortisol. Cells from cases of lepromatous leprosy show no polyclonal B-cell activation in the presence of extracts of M. leprae, although they respond normally to other mycobacterial species. The polyclonal B-cell activation response to the M. leprae extract, of a small group of normal leprosy contacts, was reduced relative to that of non-contacts.

Some suppressor cells are reported to lose their activity when precultured without stimulus in vitro. We have investigated the role of such suppressors in responsiveness to mycobacterial antigens of peripheral blood mononuclear cells (PBMNC) from patients with leprosy or tuberculosis, or from normal donors. Delayed addition of mycobacterial antigens (Mycobacterium leprae, Mycobacterium vaccae and Mycobacterium tuberculosis), but not of a fungal antigen (Candida albicans) caused enhanced responses using PBMNC from most normal donors, or tuberculoid leprosy (TT/BT) patients. However, the effect was less common using PBMNC from the lepromatous leprosy (BL/LL) group. (P less than 01.01, using M. leprae, relative to the TT/BT group), suggesting that this type of suppression reflects a normal mechanism, which is diminished rather than increased in anergic patients. Delayed addition of antigens to 'E'-rosetting cells did not result in enhanced responses. However, the different effects of 'E'-rosetting on the responses to the mycobacterial antigens of cells from normals, TT/BT and BL/LL patients, suggested that there may be two types of proliferative response to these antigens.

When cultured in 20% heat-inactivated human serum, human monocytes from seven donors were not on average significantly different from non-activated murine peritoneal cells (cultured simultaneously and in an identical manner) in their ability to inhibit BCG and, when calculated relative to growth of bacilli in the same medium without macrophages, to enhance the growth of Mycobacterium tuberculosis. Recombinant gamma-interferon caused marked inhibition of virulent M. tuberculosis by murine (BALB/c) peritoneal macrophages. This effect was seen, whether the cells were cultured in 10% fetal calf serum or in 20% heat-inactivated normal human serum, with or without the addition of iron supplements. However, unlike murine cells, the addition of crude lymphokine or recombinant gamma-interferon to human monocytes caused only weak inhibition of M. tuberculosis, and in some instances, gamma-interferon caused enhancement of growth of the bacilli. Monocytes were only slightly more effective if precultured for 4-8 days before the addition of the activating stimulus. This relative failure to develop anti-mycobacterial mechanisms occurred in spite of the activation of the cells as shown by a massive increase in reduction of nitro-blue tetrazolium inducible by phorbol myristate acetate.

Previous studies have shown that recombinant interferon gamma (IFN-gamma), crude T cell supernatants, or appropriate T-cell lines can cause total inhibition of the growth of M. tuberculosis inside murine peritoneal macrophages. In similar experiments with human monocytes much smaller effects are seen. This could be due to the relative immaturity of these cells. Because dihydroxy vitamin D3 (1,25-(OH)2 D3) can cause phenotypic differentiation of immature leukemic lines into macrophage-like cells, we have explored the possibility that exposure to cholecalciferol metabolites in vitro might increase the ability of monocytes to control proliferation of M. tuberculosis, or cause monocytes to mature into cells able to respond appropriately to IFN-gamma. Incubation of monocytes with three cholecalciferol metabolites induced anti-tuberculosis activity to an extent that correlated with their binding affinities to the intracellular receptor protein for the derivatives. 1,25-(OH)2 D3 also primed monocytes for phorbol myristate acetate-triggered reduction of nitroblue tetrazolium. The effects were additive rather than synergistic with those of IFN-gamma. Monocytes incubated with IFN-gamma developed 25-OH D3 1-hydroxylase activity, detected by conversion of tritiated 25-(OH) D3 to a more polar metabolite which coeluted with 1,25-(OH)2 D3 on straight and reverse-phase HPLC. The latter is a more active form in vivo. These findings help to explain claims for the efficacy of vitamin D in the treatment of some forms of tuberculosis, and also the occasional finding of raised serum calcium, and disturbed vitamin D metabolism in these patients.

Soluble antigen preparations from Mycobacterium leprae are reported to inhibit the response to other stimuli, of peripheral blood mononuclear cells from lepromatous leprosy cases (BL/LL) and also from tuberculoid cases (TT/BL) and normal donors. We confirm these findings and in addition, report that a similar suppressive effect is exerted by antigen from Mycobacterium vaccae, Mycobacterium nonchromogenicum and, to a lesser extent, Mycobacterium tuberculosis and Mycobactyerium kansasii. Moreover, suppression is seen using peripheral blood mononuclear cells from individuals unlikely to have encountered the organism used. The suppression is not due to toxicity of the antigen preparation, and is not indomethacin sensitive. It involves a cell found in the E-rosetting population, which loses its ability to suppress or be suppressed, after 48 hr in culture. Possible explanations include a pharmacological effect of cell wall peptidoglycolipids, or the triggering of suppressor cells specific for common mycobacterial antigens.

In order to study the antigen-presenting cell (APC) requirements for the priming of delayed-type hypersensitivity (DTH), murine spleen cells were fractionated on bovine serum albumin gradients, pulsed in vitro with tuberculin, and then injected subcutaneously into normal mice. The other footpad was challenged with tuberculin between 24 hr and 7 days later and swelling was measured 2 hr and 18-24 hr after challenge. Optimum priming for 2-hr and 24-hr responses at 7 days was achieved by an injection of 5 X 10(5) tuberculin-pulsed intermediate-density Fc + ve cells. Time-course studies revealed that the 2-hr component could be elicited as early as 24 hr after injection of pulsed APC, while the 24-hr component became significant at 3 days. Elimination of T cells or B cells did not affect the response. Injection of pulsed APC into allogeneic mice primed the 2-hr but not the 24-hr component. Neither pulsed high-density cells (mostly T cells) nor pulsed dendritic cells (DC) primed mice for these responses. Failure to elicit DTH after injection of tuberculin-pulsed DC was due to their failure to prime the 2-hr component, which other authors have shown to be a prerequisite for the appearance of the later components. That DC did prime the MHC-restricted 24-hr component was demonstrated by protocols involving the use of both macrophages and DC as APC.

Rheumatic fever is associated with exaggerated activity of B cells with massive production of antibody to the Group A streptococcus. Gc (vitamin D-binding protein) is constitutively expressed on B-cell membranes in association with membrane immunoglobulin, and could be involved in cell activation. We therefore looked for associations between the three major Gc alleles and susceptibility to rheumatic fever in a homogeneous Arab population. Patients with tuberculosis or rheumatoid arthritis and control donors, were studied in parallel. Allele frequencies in the controls, rheumatoid and tuberculosis patients were identical to those found in a previous study of normal Arab donors. However, there was a striking association between Gc2 and rheumatic fever. This allele was twice as common in these patients as in controls (p = 0.0024), and was present in 56.4% of all rheumatic fever patients.

A report is given of the use of the enzyme-linked immunosorbent assay to measure antibody to preparations of human thymocyte membranes (HTMA) and to beta 2-microglobulin. The assay described is simple and rapid, and requires only small quantities of an easily stored membrane preparation. The advantages of this technique over conventional methods involving cytotoxicity are discussed. Raised levels of IgM antibody to beta 2-microglobulin were detected in sera from SLE patients. Raised levels of IgG and IgM antibody to HTMA were found in sera from most active lepromatous cases. Two of eight sera from SLE patients showed raised IgG anti-HMTA, but not raised IgM. An attempt was made to study the subclass of the IgG antibodies found, but when checked against purified human IgG myeloma proteins, the available anti-subclass sera were found to lack the necessary degree of specificity in this assay.

Twelve preparations from mycobacterial cell walls and culture supernatant fluids were tested for their ability to activate lymphocytes from Balb/c or Nu/Nu mice, and to increase the release of mediators from macrophages in vitro. The peptidoglycolipids (wax D) were B-cell mitogens and induced plaque-forming cells. These properties were lost if the glycopeptide component was removed, leaving the pure lipid, mycolic acid. Neither the glycopeptide fractions, with well-documented adjuvant properties, nor mycobacterial polysacchardie II-activated B cells. Only intact peptidoglycolipid showed synergy with the mitogenic effect of phytohaemagglutinin (PHA) on thymocytes from Balb/c mice. The effect was much smaller than with E. coli lipopolysacharide (LPS) or dextran sulphate. The peptidoglycolipid also enhanced the release of factors from macrophages able to modify the response of Balb/c thymus cells to PHA. In this respect it resembled E. coli LPS. Adjuvant active glycopeptides did not share this property.

Samples of Bacille Calmette-Guérin (BCG) vaccines from four collaborating production laboratories, each of which had prepared vaccine from four different daughter strains of BCG, had previously been monitored for changes in colony morphology and the present study was undertaken to determine whether the changes observed were reflected in the patterns of protein secretion and lipid content. In the samples examined there was evidence for a correlation between colony morphology and the presence or absence of mycoside B. As the components of BCG that determine virulence and protective immunity are unknown, care must be taken to ensure constancy of the strains during the manufacture of vaccines.

We have previously demonstrated raised levels of IgG and IgA antibody to the mycobacterial 65-kDa heat shock protein (hsp) in the sera of patients with rheumatoid arthritis (RA). We have now attempted to determine whether this phenomenon is specific for RA, and whether it is seen only with the mycobacterial homologue of this particular hsp gene family. We therefore screened antibody levels to the mycobacterial and Escherichia coli hsp 65, and the mycobacterial, E. coli, and human hsp70, in sera from RA, systemic lupus erythematosus (SLE), tuberculosis (TB), ankylosing spondylitis (AS), Crohn's disease, and control donors. RA sera show the greatest increase in IgA binding to the mycobacterial hsp65, but no increase in IgA binding to the E. coli homologue. Similarly, only RA and TB sera show increased IgG binding to the mycobacterial hsp65, and we have shown previously that the titre is greater in RA. In contrast, the use of mycobacterial and E. coli hsp70 preparations as control bacterial hsp gene products has shown that RA patients do not differ from TB or SLE patients in their antibody binding to these proteins. Moreover, neither IgA nor IgG antibody to the human hsp70 in RA sera were higher than in TB, and the IgA binding was not higher than in SLE. These findings suggest that elevated IgG antibody levels to the mycobacterial hsp65 shows some disease specificity, and further studies with the human homologue and at the T-cell level are required.

We describe the use of 5-hydroxytryptamine (5HT) as an adjuvant in the induction of the delayed-type hypersensitivity (DTH) response to purified protein derivative (PPD). Based upon our previous studies with antigen-pulsed macrophages (M phi), we have shown that both the Day 2 early (2 hr) reaction and the Day 3 (24 hr) reaction are augmented if 5HT is incorporated into the priming injection. Furthermore, we have confirmed that in contrast to M phi, antigen-pulsed dendritic cells (DC) fail to prime the early (2 hr) component of DTH. However, DC do prime the early response if injected along with 5HT. A peripheral 5HT antagonist, ICS 205-930, inhibits both the M phi-mediated and the 5HT/DC-primed reactions. These findings support the hypothesis that DTH reactions require a cascade of both inflammatory and immunological signals, and that in mice vascular permeability mediated via 5HT is important in the early phase of the reaction.