Cortisol metabolism, cortisol sensitivity and the pathogenesis of leprosy reactions.

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.

Changes in cellular response to mycobacterial antigens and cytokine production patterns in leprosy patients during multiple drug therapy.

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.

Glycosylation of IgG during potentially arthritogenic lentiviral infections.

Agalactosyl IgG [Gal(0)] was first discovered in patients with rheumatoid arthritis (RA). However, the proportion of this glycoform is also raised in tuberculosis and leprosy. This has helped reinforce the suggestion that RA may be triggered by a mycobacterium-like slow bacterial infection. On the other hand, arthritis can occur in mycobacterial diseases, so raised Gal(0) could be associated with a tendency to arthritis, rather than with a particular type of infection. Therefore, we wished to find out whether the percentage of Gal(0) [%Gal(0)] is increased in sheep and goats following infection with maedi visna virus or caprine arthritis encephalitis virus (CAEV), both of which can lead to inflammatory synovitis. We found that the normal level of Gal(0) in these species is much lower than in humans. Goats infected with CAEV or Mycobacterium paratuberculosis (used as a control mycobacterial infection) had a significant increase in %Gal(0), though it was still below the level seen in normal humans. Studies by Western blot confirmed the presence of terminal N-acetylglucosamine on heavy chains, and percentages of Gal(0) comparable to those seen in human RA could be generated by exposing goat IgG to streptococcal beta-galactosidase. The rise in %Gal(0) was greatest in members of infected herds that were just starting to manifest arthritis, and tended to be lower in those in which severe carpitis had developed at the time of bleeding, implying the possibility that raise %Gal(0) may be an early or predisposing event for the development of arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular immune response to common mycobacterial antigens in subjects seropositive for Trypanosoma cruzi.

The immune response is impaired in the silent stage of Chagas' disease. We used quadruple skin-testing with new tuberculins in 37 adults who were symptom-free but seropositive for Trypanosoma cruzi and in 37 matched seronegative controls. Whereas 19% of controls responded to common mycobacterial antigens, none of the Chagas' seropositive group responded to them (p < 0.006), demonstrating specificity in their unresponsiveness. The enhanced tuberculin reactivity after BCG vaccination in the control group was suppressed in seropositive subjects (p < 0.002). Selective loss of response to common mycobacterial antigens may have implications for the autoimmune pathology of Chagas' disease, and for susceptibility to tuberculosis, leprosy, and HIV disease.

Identification of an antigenic domain on Mycobacterium leprae protein antigen 85B, which is specifically recognized by antibodies from patients with leprosy.

Sixty-three overlapping 15-oligomer peptides covering the 30-kDa protein antigen 85B of Mycobacterium leprae were tested by ELISA to identify epitopes recognized by human antibodies. Serum samples from patients with lepromatous leprosy (LL) reacted mainly with peptides comprising amino acid regions (AA) 206-230, 251-280, and 291-325. Sera of patients with active tuberculosis who responded to the native 30-kDa antigen did not recognize these peptides. The antibody-binding specificity to the defined B cell regions was evaluated in a blind study with 71 serum samples from patients and household contacts living in Ethiopia where leprosy is endemic. The peptide of AA 256-280 was recognized by 88% of LL patients, 15% of patients with tuberculoid leprosy, and none of the contacts. These findings suggest that there are major linear B cell epitopes on the M. leprae 30-kDa protein that are recognized by lepromin-negative LL patients, whereas lepromin-positive patients respond preferentially to conformational epitopes.

Slow bacterial infections or autoimmunity?

In this article, Graham Rook and John Stanford propose that a group of idiopathic diseases that are often associated with a degree of autoimmunity and arthritis, including rheumatoid arthritis, inflammatory bowel disease, sarcoidosis and psoriasis, are caused by extremely slow-growing bacteria. They suggest that these diseases are one end of a continuous spectrum caused by related slow-growing genera, which ranges from rheumatoid arthritis, through Takayasu's arteritis and Whipple's disease, to reach the conventional mycobacterioses such as tuberculosis and leprosy.

Effect of mycobacteria on sensitivity to the cytotoxic effects of tumor necrosis factor.

Unlike Mycobacterium leprae, Mycobacterium tuberculosis is not found inside cells other than macrophages and polymorphonuclear cells in vivo, yet previous work has revealed that in vitro it readily enters all cell lines tested. Moreover, these cells are not killed by the intracellular mycobacteria. We report here that when fibroblasts take up live (but not killed) M. tuberculosis H37Rv, they develop greatly increased sensitivity to the toxic effects of tumor necrosis factor (TNF) whether the cell line is inherently sensitive to TNF or not. Ultrasonically disrupted M. tuberculosis also has this property. The increased sensitivity is seen in the absence of metabolic inhibitors, although addition of emetine, an inhibitor of protein synthesis, causes the effect to manifest itself earlier and at a lower concentration of TNF. In contrast, infection with Mycobacterium bovis bacillus Calmette-Guérin induces little or no increased sensitivity to TNF, whereas Mycobacterium avium and M. tuberculosis H37Ra have intermediate sensitivities. We discuss the possibility that virulent tuberculosis strains produce a factor which distorts the normal protective function of TNF, rendering it toxic to host tissues and leading to the classical immunopathology of tuberculous lesions.

Cytokines and the Koch phenomenon.

We outline the mechanisms contributing to the human form of the Koch phenomenon, which we define as necrosis occurring within 24-48 h of injection of mycobacterial antigen into the skin of past or present tuberculosis patients. It is probable that tissue damage mediated in the same way occurs in the lesions themselves. We suggest that the necrosis is mediated in part by cytokines, particularly Tumour Necrosis Factor (TNF), and that this occurs for three reasons. First, Mycobacterium tuberculosis evokes an immunoregulatory abnormality characterised by raised agalactosyl IgG. This abnormality, also found in rheumatoid arthritis, Crohn's disease, and Erythema Nodosum Leprosum, seems to be associated with dysregulation of cytokine release. Secondly, M. tuberculosis itself triggers further cytokine release. Thirdly, the normally protective role of TNF is distorted by several interacting properties of components of M. tuberculosis, which render the cytokine toxic to the host tissues. The immunoregulatory abnormality may be susceptible to correction by immunotherapy.

Mycobacterium vaccae in immunoprophylaxis and immunotherapy of leprosy and tuberculosis.

Both leprosy and tuberculosis present continuing problems in their control, especially in the developing world, despite the availability of drugs effective in producing a bacteriological cure. Improved immunoprophylaxis, and an effective immunotherapy to be used with chemotherapy are urgently required. Intradermal injection of a suspension of killed Mycobacterium vaccae promotes cell-mediated responses to antigens common to all mycobacteria, and switches off the tissue-necrotizing aspects of the Koch phenomenon. These properties led to the use of the suspensions as an improved vaccine, either alone or in combination with BCG. The same properties led to the employment of the suspension in immunotherapy as an adjunct to chemotherapy in the treatment of both leprosy and tuberculosis. The evidence leading to these conclusions is reviewed and discussed.

Immunotherapy with Mycobacterium vaccae as an adjunct to chemotherapy in the treatment of pulmonary tuberculosis.

47 patients with adult-type pulmonary tuberculosis attending the Chest Diseases Hospital in Kuwait were given a single injection of 10(9) irradiation-killed M. vaccae after 1 month of a 9-month course of chemotherapy. The patients were followed-up for 3 more months in double blind comparison with 65 patients given an injection of saline (placebo). The immunotherapeutic injection produced a small local lesion in 44/47 patients, 18 of which ulcerated and produced small scars. Immunotherapy made no measurable difference to the bacteriological, biochemical, haematological, or radiological parameters measured. However it was associated with significantly improved weight gain, reduced size of skin test response to Tuberculin, increased lymphocyte proliferation to common mycobacterial antigens, and increased antibody levels to mycobacterial antigens. The changes in skin test and LTT responses were related and occurred in 29% of patients whose recognition of common mycobacterial antigens returned to normal. The remaining patients did not differ in these respects from those receiving placebo. The proportion of patients whose responses were improved was very similar to that achieved using the same immunotherapeutic agent in a group of treated multibacillary leprosy patients.

Agalactosyl IgG in inflammatory bowel disease: correlation with C-reactive protein.

The proportion of oligosaccharide chains on the Fc fragment of IgG which terminate with N-acetylglucosamine (GlcNAc) rather than galactose is increased in rheumatoid arthritis and tuberculosis, and in sera from patients with Crohn's disease, probably because of decreased activity of a galactosyltransferase in B lymphocytes. We have assayed the prevalence of agalactosyl oligosaccharides on IgG in sera from 67 patients with inflammatory bowel disease (32 ulcerative colitis and 35 Crohn's disease). The prevalence of agalactosyl IgG significantly increases in the majority of Crohn's patients (19/35 patients), and correlates with the level of C-reactive protein (r = 0.79), and inversely with the concentration of serum albumin. Sera from ulcerative colitis patients show less frequent (nine of 32) and less marked rises in agalactosyl IgG, and sera with high C-reactive protein values can contain normal levels. Thus in ulcerative colitis no correlation was seen between the two assays. The diseases in which the percentage of agalactosyl IgG is raised (rheumatoid arthritis, tuberculosis, Crohn's disease and some ulcerative colitis) are characterised by simultaneous T cell mediated granulomatous tissue damage, and acute phase responses. Levels are normal in less tissue damaging granulomatous conditions, including sarcoidosis, and leprosy (except during episodes of erythema nodosum leprosum). We suggest therefore that a raised percentage of agalactosyl IgG is a correlate of a particular type of T cell mediated pathology which may be relevant to the pathogenesis of inflammatory bowel disease.

A transient rise in agalactosyl IgG correlating with free interleukin 2 receptors, during episodes of erythema nodosum leprosum.

The proportion of oligosaccharide chains on the Fc fragment of IgG which terminate with N-acetylglucosamine and not galactose (%GO) has previously been shown to be raised in rheumatoid arthritis (RA), Crohn's disease (CD) and tuberculosis (Tb), but to be normal in sarcoidosis (SA), and in both lepromatous and tuberculoid leprosy. However we have now studied %GO in sequential serum samples collected from lepromatous leprosy patients undergoing episodes of erythema nodosum leprosum (ENL). During ENL %GO is transiently raised, and this rise parallels an increase in circulating interleukin 2 receptors (IL-2R). These findings confirm that changes in T cell function occur during ENL. Moreover it appears that %GO rises when there is, simultaneously, T-cell-mediated tissue damage and an acute phase response (RA, CD, Tb, ENL), but not when there is an acute phase response without major T cell involvement, or chronic T cell activity alone (SA, and tuberculoid leprosy). We suggest therefore that %GO is an indicator of a type of T cell activity with broad immunopathological implications.

Vaccination and skin test studies on children living in villages with differing endemicity for leprosy and tuberculosis.

The purpose of this study carried out in Iranian Azerbaijan was to determine the pattern of skin-test positivity to mycobacterial antigens in children living in the valley, and to assess the effect on this of a series of vaccines against mycobacterial disease. Set up in 1978, 1707 tuberculin-negative children without scars of previous BCG vaccination were vaccinated with BCG Glaxo alone (vaccine A) or with the addition of a suspension of killed Mycobacterium vaccae (vaccine B). One hundred children were vaccinated with BCG Glaxo plus a suspension of M. leprae (vaccine C). Eight to 10 years later about half of the children were found for follow up. At this time further children were skin tested, and the results obtained were related to whether or not they had scars of vaccination with BCG Pasteur (Teheran) given by the local health authorities. Between setting up the study and the first follow up, cases of leprosy or tuberculosis had occurred in some of the villages, although not among those we had vaccinated. Differences between the effects of the vaccines were only found in villages with cases of leprosy. In these villages positivity to leprosin A was significantly greater after vaccine B (49%) than after vaccine A (36%; p less than 0.04). The results for scrofulin and vaccine were the same after both vaccines, and significantly lower than in the villages without cases of leprosy. The general reduction in skin-test positivity in the villages with leprosy cases was mainly due to a loss of category 1 responders to group i, common mycobacterial, antigens. It was concluded that where casual contact with cases of leprosy occurs the combination of BCG with killed M. vaccae is likely to be a better vaccine for leprosy than is BCG alone. Although few children received the combination with M. leprae, the results obtained were not particularly promising.

The role of cytokines in the immunopathology of tuberculosis, and the regulation of agalactosyl IgG.

Tuberculosis is characterised by necrosis in the lesions and in skin-test sites, and by fever and weight loss. In contrast, other diseases with chronic T cell mediated responses, such as uncomplicated leprosy and sarcoidosis, have non-necrotising lesions with little systemic upset. Crude sonicates of M. tuberculosis and M. leprae prepare skin sites for TNF-mediated damage via a pathway which unexpectedly appears to involve CD8+ T cells, and both mycobacteria contain potent triggers of TNF release (lipoarabinomannan and peptidoglycan derivatives). These observations can partially explain the pathology of tuberculosis, but fail to explain why similar events do not normally occur in leprosy. It now seems likely that the answer lies in the existence of novel regulatory pathways. A recently recognised correlate (or consequence) of diseases characterised by T cell-dependent tissue-damaging pathology and cytokine release, is an increase in the level of agalactosyl IgG. This behaves like a T cell-dependent acute phase reactant, and is raised in tuberculosis, rheumatoid arthritis, and Crohn's disease, but not in sarcoidosis or uncomplicated leprosy. Thus it may act as a marker for a type of pathology of very broad significance, though its functional role remains obscure.

Unexpected findings amongst the skin test responses to mycobacteria of BCG vaccinated Kuwaiti school children.

A multiple skin test survey was carried out in Kuwait on 1200 school children aged 8-11 years, and on 1228 children aged 12-16 years. With only 15 exceptions, all these children had received vaccination with Japanese BCG just before they started school, 5 years and 9 years earlier respectively. Tuberculin positivity was almost 90% in both groups, with a mean response size of 8.7 mm. This was associated with remarkably high responsiveness to many of the other mycobacterial species investigated. Since this high reactivity was also to Mycobacterium ulcerans, a species most unlikely to be present in Kuwait, it is proposed that this might be due to responsiveness to group ii antigen which is present in all slow growing species. Only M. flavescens and M. rhodesiae amongst the fast growing species, were absent as sensitising organisms. After correction for the supposed reactivity to group ii antigen, M. avium B, M. gordonae, M. ulcerans and M. xenopi amongst the slow growing species, also appeared to be absent from the Kuwait environment. The species most commonly encountered were M. leprae, M. chitae, M. neoaurum, M. diernhoferi, and M. vaccae in this order. This was a remarkable finding for a country assumed to be poor in contact with environmental species, and known to have a very low prevalence of leprosy. As previously reported from Iran, but not confirmed in other places, there was a 95% correlation between responsiveness to Leprosin A and Vaccin. Amongst the slow growing species M. avium A, M. intracellulare, and M. kansasii appear to be frequent sensitising agents, in common with many other places.

A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles.

The individual antigenic components present in microgram quantities of complex mixtures can be separated reproducibly by polyacrylamide gel electrophoresis and transferred onto nitrocellulose blots. We report that the ng quantities of antigen present in single lines cut from such Western blots can be used to induce maximal lymphoproliferative responses in 30-60 microtitre wells. In order to achieve this the excised lines of antigen-bearing nitrocellulose sheet must be converted into antigen-bearing particles small enough to be engulfed by macrophages. We describe optimal conditions and discuss the applications of this technique.

Skin sensitization to mycobacteria amongst school children prior to a study of BCG vaccination in North Lebanon.

1888 school children aged between 7 and 17 years, living in 13 villages in two districts of North Lebanon, were skin tested with four new tuberculins as the initial step in a study of BCG vaccination. The great majority of children were tested with Tuberculin, Leprosin A, Vaccin and Scrofulin. In comparison with other countries where similar studies have been carried out, extremely low levels of sensitization were discovered, indicating very little contact with mycobacteria. There was, however, a statistically significant increase in positivity with increasing age. The results obtained for the villages of each district were significantly different from each other, positivity being greatest in Akkar district for each reagent. The eight villages of Zgharta district could be separated into a lowland group of four villages, a mountain group of three villages and one anomalous mountain village. There was significantly more positivity in the lowland than in the mountain villages. In Akkar district, where leprosy has a low prevalence, positivity to Leprosin A was 8% amongst the children (leaving out an anomalous village). In Zgharta district where the disease does not occur, positivity was 3.4% to Leprosin A for the lowland villages and 0.9% for the group of 3 mountain villages. The two anomalous villages were the only ones in which tuberculosis cases were known to have occurred recently, and they were the only two villages in which Tuberculin positivity exceeded 10%.