High dose prednisolone treatment of leprosy patients undergoing reactions is associated with a rapid decrease in urinary nitric oxide metabolites and clinical improvement.

Evidence is accumulating that nitric oxide (NO) produced by macrophages has a role in the pathogenesis of reactions in leprosy. We followed the urinary levels of the metabolites of NO [nitrite (NO2-) and nitrate (NO3-)] and the clinical response to prednisolone treatment in leprosy patients (n = 9) admitted to ALERT leprosy hospital Addis Ababa, Ethiopia, because of reversal reaction (RR) or erythema nodosum leprosum (ENL). In untreated reactional leprosy patients, the levels of urinary NO metabolites (1645 +/- 454 microM, n = 9, ENL = 4, RR = 5) decreased significantly 2 weeks after high dose prednisolone treatment (1075 +/- 414 microM, P < 0.05), and remained stable 4 (895 +/- 385 microM, P < 0.02) and 6 weeks following treatment initiation (1048 +/- 452 microM, P < 0.02). This decrease was also present when the reactional patients were subdivided according to the type of reaction (ENL, RR) and coincided with a clinical improvement. In patients showing a poor clinical response to steroids, no or minor effects on the urinary NO metabolite levels were observed. We conclude that there is a correlation between the decrease in urinary NO metabolites and a favourable clinical response after high dose prednisolone treatment of reactional leprosy patients.

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

Diagnostic assays for leprosy based on T-cell epitopes.

To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.

Effects of HIV co-infection and chemotherapy on the urinary levels of nitric oxide metabolites in patients with pulmonary tuberculosis.

The presence of nitric oxide (NO) and its role as a factor in host defence against intracellular pathogens in human macrophages is controversial. We measured the metabolites of NO (nitrite (NO2-) and nitrate (NO3-)) in urine from Ethiopian patients suffering from tuberculosis. The urinary level of NO2-/NO3- in a group of healthy Ethiopians was 1020+/-471 microM (n = 22). Untreated HIV negative patients with active pulmonary tuberculosis (1574+/-588 microM, p<0.01, n = 12) and household contacts to tuberculosis patients (1949+/-812 microM, p = 0.006, n = 7) had significantly higher levels of urinary NO2-/NO3- than the control group. Untreated HIV positive patients with pulmonary tuberculosis did not have increased levels of urinary NO2-/NO3- (1101+/-614 microM, n = 6). Some of the HIV negative untreated patients with pulmonary tuberculosis (1710+/-519 microM, n = 6) were followed up after treatment and showed a reduction in the levels of urinary NO2-/NO3- 1 week after treatment (945+/-599 microM, p<0.05). We conclude that HIV negative patients with active pulmonary tuberculosis have increased urinary levels of nitric oxide metabolites with a reduction following specific anti-tuberculous chemotherapy.

Increased levels of nitric oxide metabolites in urine from leprosy patients in reversal reaction.

We measured the metabolites of NO [nitrite (NO2-) and nitrate (NO3-)] in urine from Ethiopian patients suffering from leprosy. The urinary level of NO2-/NO3- in a group of healthy Ethiopians was 1020 +/- 471 microM (n = 22). Leprosy patients in reversal reaction had significantly higher levels of NO2-/NO3- (1817 +/- 492 microM, p < 0.001, n = 12) than both the control group and leprosy patients who were not in reversal reaction (1079 +/- 446 microM, n = 12). We conclude that the reversal reaction in leprosy in associated with increased urinary levels of nitric oxide metabolites.

Improved microscopical diagnosis of pulmonary tuberculosis in developing countries.

The diagnosis of pulmonary tuberculosis (TB) relies on the bacteriological examination of sputum. However, microscopy of smears made directly from sputum has a low sensitivity and there is an urgent need for improved methods. We have compared microscopy of smears made directly from sputum with microscopy after liquefaction of sputum with household bleach (NaOCl) and concentration of bacteria by centrifugation. In 3 studies performed in Ethiopia and India, the use of the NaOCl method increased the number of samples positive for acid-fast bacilli by more than 100%. The technique is appropriate for developing countries and its application would increase the efficiency of TB control programmes. As a potent disinfectant, NaOCl also has the advantage of lowering the risk of laboratory infection.

The response of human B cells to Mycobacterium leprae. Identification of target antigens following polyclonal activation in vitro.

We have investigated the B cell response to Mycobacterium leprae in leprosy patients and healthy controls. A comparison of Western-blotted proteins separated by two-dimensional gel electrophoresis and probed with pooled sera from LL and BT patients revealed distinct antigen recognition patterns for the two classifications of the disease. To characterize the circulating B cells capable of producing anti-M. leprae antibodies in vitro, peripheral blood lymphocyte cultures were activated polyclonally with an anti-CD3 mAb. The resulting culture supernatants were used to probe Western-blotted M.leprae proteins and contained antibody reactive with a 10 kd M.leprae antigen. This antibody was absent in stimulated culture supernatants from healthy occupational contacts or unexposed controls, suggesting the specificity of the response. Distinct repertoires of serum and culture supernatant anti-M.leprae antibodies were observed when Western-blotted antigens were probed after two-dimensional gel electrophoresis. This method for assay of specific antibody production against individual components present in a complex mixture of antigens after polyclonal activation in vitro may be used to study the regulation of B cell activation in leprosy and other diseases.

T cell responses to fractionated Mycobacterium leprae antigens in leprosy. The lepromatous nonresponder defect can be overcome in vitro by stimulation with fractionated M. leprae components.

Protective immunity against Mycobacterium leprae is dependent on M. leprae-reactive T lymphocytes. M. lepare-directed T cell reactivity is high in the localized tuberculoid form of leprosy but specifically absent in the disseminated lepromatous type of the disease. Two important questions that are relevant for the understanding of the immune response in leprosy as well as for the design of rational immunoprophylaxis and -therapy strategies are: (a) what are the antigens that trigger T cell responses in tuberculoid patients and thus protect these individuals from developing lepromatous leprosy and (b) is it possible to restore T cell responsiveness to M. leprae in lepromatous patients by rechallenging the immune system with selected antigens that will trigger help but not suppression? We have addressed these question by directly probing the peripheral T cell repertoire of 10 tuberculoid and 18 lepromatous patients with large numbers of different M. leprae and BCG antigenic components that had been separated on the basis of their relative molecular mass (Mr) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose. This technique allows the identification of T cell-stimulating antigens independent of the expression of B cell epitopes by these antigens. So far T cell epitopes have only been mapped on M. leprae proteins that had previously been defined by antibodies. Our results show that: (a) tuberculoid patients' T cells responded preferentially to M. leprae and BCG antigens in the lower (i.e. less than 70 kDa) Mr range with a peak in the 10-25 kDa range; (b) 6 out of 18 lepromatous patients that did not respond to whole M. leprae responded strongly to isolated M. leprae components; antigens in the lower Mr. range were recognized by five out of these six patients and thus commonly seen by both tuberculoid and lepromatous patients' T cells; however, antigens in the higher Mr range, in particular greater than 150 kDa, were only recognized by lepromatous patients' T lymphocytes; (c) furthermore, the T and B cell repertoires in leprosy patients are skewed towards different antigenic fractions.

Cellular, humoral, and gamma interferon responses to Mycobacterium leprae and BCG antigens in healthy individuals exposed to leprosy.

Protective immunity against mycobacteria is dependent on antigen-specific T cells. The antibodies induced upon immunization with mycobacteria have no apparent role in host protection. Serological techniques have detected some antigens that are also recognized by human T cells but may fail to recognize others. Potentially, there may be differences in the epitopes seen by the T and B cell anti-mycobacterial antigen repertoires. We have screened the different components of sonicated BCG or Mycobacterium leprae that were separated according to their molecular weight (MW) by SDS-PAGE and then electroblotted on nitrocellulose paper. The blots were cut into squares and tested directly in a T cell proliferation assay. Our results indicate that peripheral T cells of healthy leprosy patient contacts respond preferentially to the lower MW (less than 70,000) and not the higher MW fractions of M. leprae and BCG, in contrast to the humoral response of these same individuals. The most important fractions in inducing a lymphoproliferative response were in the regions of 11-16 kDa of BCG and M. leprae and to the 22-26 kDa region of M. leprae. These fractions appeared to represent molecular weight regions that were in some instances clearly distinct from previously defined antigens. It was further shown that lymphoproliferation in response to mycobacterial fractions correlated with the production of gamma interferon, a lymphokine required for macrophage activation and elimination of mycobacteria. These studies allow the direct assessment of antigens involved in protective T cell-mediated immunity, and should be helpful in selecting relevant antigens for skin testing and immunization.

Analysis of the antigenic profile of Mycobacterium leprae: cross-reactive and unique specificities of human and rabbit antibodies.

Thirty-two mycobacterial components were detected by antibodies contained in leprosy patients' sera across the clinical spectrum and rabbit anti-M. leprae hyperimmune sera by western blot analysis of armadillo-derived M. leprae antigen preparations. Sera of borderline tuberculoid patients were found to contain antibodies recognizing 18 M. leprae components. While the reactivity of the sera on the lepromatous pole seemed to be distributed over the entire molecular weight range, most of the reactivity in the borderline tuberculoid patients was directed at higher molecular weight components (greater than 70,000). Identification of a series of previously unrecognized M. leprae components offers new possibilities in regard to the potential use of these antigens as targets for immunodiagnosis. Antibodies contained in the rabbit anti-M. leprae sera reacted with 19 M. leprae components. Antigens migrating at 64,000, 38,000, and 22,000 were detected by the rabbit sera only. Evidence of extensive cross-reactivity between M. leprae and BCG organisms emphasizes the need to use well-characterized antibody probes to determine the specificity of select mycobacterial antigens. The potential usefulness of rabbit monospecific hyperimmune sera to select M. leprae fractions in immunodiagnosis, in immune regulation studies, or as a tool to screen for mycobacterial products in lambda gt11 phage lysates of E. coli is discussed. Select M. leprae components were partially purified and their recovery assessed through SDS-PAGE analysis of Coomassie blue-stained gels.