Leishmania in the Old World: 3. The distribution of L. aethiopica zymodemes.

Isoenzyme profiles of 28 stocks of Leishmania aethiopica were compared with those of reference strains of L. aethiopica, L. tropica and L. major using starch-gel electrophoresis of 13 enzymes (GPI, GD, ES, PGM, PEPD, NH, ASAT, ALAT, PK, MPI, 6PGD, SOD, MDH). 13 zymodemes were seen. L. aethiopica showed some infraspecific variation. Stocks from Phlebotomus longipes and Procavia habessinica were indistinguishable from those from man. Stocks from cases of diffuse cutaneous and cutaneous leishmaniasis were indistinguishable. Only one enzyme pattern was held in common with the L. tropica reference strain enzyme profile, and none with the L. major reference strain. The status of L. aethiopica as a separate species is supported.

Muramidase (lysozyme) findings in sural and radial nerve biopsies in leprosy patients after varying periods of treatment.

Using the immunoperoxidase staining method, tissue muramidase (lysozyme) activity was studied in 34 nerve biopsies from leprosy patients and compared to findings in the skin. In a majority of lepromatous and borderline-lepromatous leprosy patients, the enzyme was seen to form a saccular pattern within the cells; whereas a granular pattern was found at the tuberculoid end of the leprosy spectrum, as well as during reversal reactions. Indeed, the most intense enzymatic activity was found in four patients with reversal reactions. Compared to the skin, muramidase activity was found to be more intense and persisted longer in the nerves. Successful antileprosy treatment reduced the enzymatic activity in both the nerves and the skin, but more so in the skin. Schwann cells and axons did not show muramidase activity, indicating that the muramidase-positive cells are not of neuronal origin. Our results suggest that a high percentage of mononuclear cells infiltrating the peripheral nerves in leprosy are derived from blood monocytes. The function of tissue muramidase in leprosy is not yet clear. Its peculiar intracellular distribution pattern in the different forms of leprosy, however, warrants further study to elucidate its role in the pathogenesis of the disease.

Anti-Mycobacterium leprae antibodies in urine from lepromatous patients examined by crossed immunoelectrophoresis and radioimmunoassay.

Precipitating anti-Mycobacterium leprae antibodies were found in concentrated urine samples from 21 out of 42 lepromatous patients. These antibodies were directed against M. leprae antigens 5, 6, and 7. In a radioimmunoassay for anti-M. leprae antibodies, 90% of these patients had higher antibody levels in their urine than control persons. There was a positive correlation between anti-M. leprae antibody levels in serum and urine. The advantages of using atraumatically collected samples like urine in epidemiological work are pointed out. The present report shows that urine can be used to measure the antibody response to a specified microorganism causing infection outside the urinary tract. The possible presence of antibodies in urine should alert researchers who look for antigens in urine to choose assays that minimize interference by such antibodies.

Demonstration of mycobacterial antigens in nerve biopsies from leprosy patients using peroxidase-antiperoxidase immunoenzyme technique.

Peripheral nerve biopsies from patients with leprosy were stained with anti-Mycobacterium bovis (BCG) in a peroxidase-antiperoxidase (PAP) system to demonstrate intraneural mycobacterial antigens. Most M. leprae antigens have been shown to cross-react with BCG. Of the 30 biopsies from borderline tuberculoid (BT) patients 18 had acid-fast bacilli while 26 of them had demonstrable mycobacterial antigens in their nerves. All borderline lepromatous (BL) and lepromatous leprosy (LL) nerve biopsies had both M. leprae and mycobacterial antigens within them. Most of the antigens in the BT patients were seen to be extracellular. In BL and LL patients antigens were seen both extracellularly and intracellularly in Schwann cells and infiltrating macrophages. Mycobacterial antigens in BT nerves were always seen to be surrounded by a mononuclear cell reaction while in the BL and LL patients antigens could be seen with minimal cellular infiltrate and the neural architecture was more or less preserved. While bacilli could not be seen in BT patients who had been released from treatment for more than 4 years, mycobacterial antigens could still be seen in some patients who had been released from treatment for up to 5 years. Patients with no skin lesions but with large, painful, or tender nerves were found to have intraneural inflammation surrounding mycobacterial antigens, while those with a similar clinical picture but without tender or painful nerves showed no marked inflammation within their nerves despite the presence of mycobacterial antigens. From these findings it was concluded that immunologically mediated inflammatory response toward intraneurally located M. leprae antigens in conjunction with other host factors may be necessary for nerve damage in the BT leprosy patients. In the BL and LL patients the mechanisms of nerve damage are still unknown with certainty but local effects and immune-complex damage secondary to abundant M. leprae antigens are worth exploring. The use of immunohistological techniques should offer a new approach in the study of the immunopathology of leprosy.

Relation between anti-Mycobacterium leprae antibody activity and clinical features in borderline tuberculoid (BT) leprosy.

Antibody activity against Mycobacterium leprae antigen 7 was determined by radioimmunoassay and IgG antibodies against various antigens present in an M. leprae sonicate by a solid phase radioimmunoassay in 77 patients with borderline tuberculoid (BT) leprosy. In both assays there was a wide variation in antibody activity in individual patients although all were diagnosed as having BT leprosy. The median antibody activity was lower in newly diagnosed cases than in patients appearing with active skin lesions or new skin lesions despite dapsone (DDS) treatment of long duration. Further comparison of patients with high and low antibody activity revealed that high antibody activity was significantly correlated statistically with active skin lesions, new skin lesions and neuritis despite DDS treatment of long duration. The reason for variation in antibody activity in newly diagnosed BT leprosy remains unclear, and this patient group is of particular interest for further characterization of the basis for variation in antibody activity in tuberculoid leprosy.

Effect of treatment on antibody activity against Mycobacterium leprae antigen 7 in tuberculoid leprosy.

Anti-Mycobacterium leprae antigen 7 antibody activity was determined by radioimmunoassay during treatment in a longtime study of individual patients with newly diagnosed borderline tuberculoid (BT) leprosy and in BT leprosy patients who were suspected from their case histories to have dapsone (DDS) resistant leprosy. There was a strong correlation between clinical and antibody activity, and clinical improvement following treatment led to a marked decrease in antibody activity in most cases. A characteristic pattern of rapid and marked increase in antibody activity shortly after the initiation of treatment was observed in patients with newly diagnosed BT leprosy. This pattern may become of practical importance in the evaluation of patients with BT leprosy as an indicator that the therapy is effective, even though this pattern was associated with a transient increase in inflammatory activity in the skin lesions. The association of inflammatory activity with increased antibody activity strongly indicates that the underlying processes are associated with the stimulation of both humoral and cellular immune responses.

The role of products of the human HLA-DR locus (Ia molecules) in in vitro M. leprae driven lymphoproliferation.

We analysed the role of Ia molecules in T cell activation with M. leprae by using two hybridoma monoclonal antibodies D1-12 and D4-22 with specificity for non-polymorphic isotypes NG-1 and NG-2 respectively on the HLR-DR molecular framework. The results show that the addition of either monoclonal antibody up to 3 days after onset of culture significantly blocks the in vitro M. leprae driven lymphoproliferation. The mechanism of suppression appears to be due to a combination of steric hindrance and a vigorous suppressor signal from anti-Ia treated macrophages. The possible implications of these results are discussed.

T-cell conditioned media reverse T-cell unresponsiveness in lepromatous leprosy.

In some subjects the infective agent of leprosy, Mycobacterium leprae, causes disseminated (lepromatous) disease. Such subjects have a major role in the transmission of the disease and show deficient T-cell responses both in vivo and in vitro to M. leprae, but not to other antigens. Numerous studies have recently shown that T cells with functional capabilities after initial triggering with antigen can be maintained in a state of continuous proliferation in vitro when cultured in medium containing interleukin 2 (IL-2). Here we have studied the effect of IL-2 rich T-cell conditioned medium on lepromatous peripheral blood mononuclear cells. Our results show that although lepromatous T cells fail to produce IL-2 after exposure to M. leprae they can respond by proliferation to M. leprae in the presence of T-cell conditioned medium, suggesting that the unresponsiveness in lepromatous leprosy results from a deficiency in the production of IL-2 or related factors and not a lack of M. leprae-reactive T cells.

Nerve damage following intraneural injection of Mycobacterium leprae into rabbits pre-sensitized to mycobacteria.

Nerve damage is a common feature of leprosy although the mechanism responsible for the damage is not clearly understood. In the tuberculoid end of the leprosy spectrum where both intraneural Mycobacterium leprae or their antigens and cell-mediated hypersensitivity to M. leprae co-exist, acute neuritis affecting major nerve trunks can occur during reversal reactions. These reactions are known to be associated with increased hypersensitivity to M. leprae antigens. The nerve involvement is therefore thought to be a direct consequence of the patient's hypersensitivity to M. leprae. So far the only indirect evidence based on in vitro studies have been produced to support such a contention. We sensitized rabbits with M. leprae and then injected M. leprae sonicate into the sciatic nerves at the peak of hypersensitivity. Seventy-two hours later, the nerves were dissected out and studied histologically. Our results show that cellular infiltration and axonal degeneration can occur as a direct consequence of hypersensitivity to intraneural M. leprae antigens. This study, therefore, offers direct evidence for the involvement of specific cell-mediated hypersensitivity to M. leprae antigens in the pathogenesis of major nerve trunk damage in the tuberculoid end of the leprosy spectrum especially during acute reversal reactions.

Immunoglobulin A (IgA) in nasal washings and saliva of leprosy patients.

IgA levels in nasal washings and saliva were determined in leprosy patients and healthy controls. In addition, serum levels of IgA, IgG, and IgM and total serum protein were analyzed. IgA levels in the nasal washings, but not in the salivas, were elevated significantly in the borderline (BL) and lepromatous (LL) groups. In the tuberculoid (TT) and borderline tuberculoid (BT) groups, the IgA levels in both the nasal washings and salivas did not differ from the controls. Total protein was also elevated in the nasal washings in the BL and LL groups. Thus, the IgA expresses as mg/mg protein did not differ significantly from the control group. However, in individual patients the levels of IgA and total protein were not correlated in either the nasal washings or the salivas. The high serum immunoglobulin and protein levels were in accord with the findings of most other workers.

Immunohistological studies of skin biopsies from patients with lepromatous leprosy.

Forty-six skin biopsies from lepromatous leprosy patients were examined for immunoglobulin and complement deposits as well as mycobacterial antigens. Rabbit anti-human immunoglobulin, rabbit anti-human C3, and rabbit anti-Mycobacterium bovis (BCG) were used as the primary antigen-detecting antibodies in a peroxidase antiperoxidase technique. Of the 26 biopsies from active erythema nodosum leprosum lesions, 6 were positive for immunoglobulin or complement deposits. These deposits were found in the dermoepidermal junction, within the foamy cells, and, in one patient, around a blood vessel. Five of twenty patients with lepromatous leprosy without erythema nodosum leprosum showed similar deposits in the dermoepidermal junction and within foamy cells. None of these patients had these deposits around blood vessels. Mycobacterial antigens were seen in all biopsies studied. The presence of acute inflammatory infiltrates did not correlate with the presence or absence of immunoglobulin or complement deposits. It is felt that immunoglobulin or complement deposits are not a constant feature of early erythema nodosum leprosum lesions and that these deposits may be secondary rather than primary in these lesions.