Differing antibody IgG isotypes in the polar forms of leprosy and cutaneous leishmaniasis characterized by antigen-specific T cell anergy.

Leprosy and American cutaneous leishmaniasis are tropical diseases which present a spectrum of clinical and immunological manifestations. Lepromatous leprosy and diffuse cutaneous leishmaniasis are the severe, progressive polar forms of disease characterized by persistent T cell anergy. Relative concentrations of antibodies belonging to the four IgG isotypes have been determined in these forms of disease as well as active visceral leishmaniasis, which presents transitory T cell anergy. Leishmania-specific IgG4 antibodies predominated in 19/20 sera from patients with diffuse cutaneous leishmaniasis, and IgG1 antibodies predominated in 9/10 cases of untreated visceral leishmaniasis. The predominant IgG isotype of Mycobacterium leprae-specific antibodies in untreated lepromatous leprosy was remarkably variable (IgG1, IgG2, IgG3 and IgG4 in 8, 6, 2 and 1 sera, respectively). Differing IgG antibody isotypes have been associated with distinct CD4+ T cell helper subpopulations and their characteristic lymphokine profiles in several pathologies. These results suggest that T cell anergy in chronic intracellular infections may be associated with as yet undefined mechanisms which modulate reported T helper cell-lymphokine isotype relationships.     

Characterization of the cellular immune defect in lepromatous leprosy: a specific lack of circulating mycobacterium leprae-reactive lymphocytes

The blastogenic response of leucocyte cultures from patients with tuberculoid and lepromatous leprosy has been studied. The leucocytes from the two groups were studied simultaneously and cultivated in the same pool of normal human serum. While the leucocytes from twenty-eight tuberculoid patients responded quite strongly to Mycobacterium leprae after 7 days of culture (average lymphocyte transformation 11·1%), there was a complete lack of response in similar cultures from twenty-seven lepromatous patients (average 0·1% transformed cells). These results were confirmed by studies on cellular incorporation of ³H-thymidine in the cultures from four tuberculoid and four lepromatous patients.

This lack of response was quite specific as leucocytes from several lepromatous patients responded to BCG. Furthermore, four patients with both lepromatous leprosy and tuberculosis responded as strongly to BCG and PPD as tuberculous patients without leprosy. In the mixed leucocyte reaction, between two lepromatous or two tuberculoid patients respectively, the lepromatous cells responded well (average 15·0%) and comparably to tuberculoid cells (average 12·1%).

The blastogenic response of purified lymphocytes to M. leprae revealed a similar pattern, i.e. the tuberculoid cells responded well, while again there was a lack of response in the lepromatous group.

It is concluded that the lepromatous patients lack circulating lymphocytes responding to M. leprae, indicating that their immunological defect as observed in the present study has features in common with immunological tolerance.

     

Experimental Murine Leprosy

Sera from C3H and C57BL mice infected with Mycobacterium lepraemurium (MLM) and from human leprosy patients were examined for antibodies against MLM by a crossed Immunoelectrophoresis (CIE) technique. Altogether antibodies against six or seven MLM antigens were found in the mouse sera. After a small inoculum of MLM, C3H mice produced more antibodies than C57BL mice After a large inoculum both strains produced about the same amount of antibodies but showed qualitative differences in their response patterns. A serum pool from patients with lepromatous leprosy contained antibodies against six MLM antigens: five of these were identical with the antigens against which antibodies were found in infected mice. C57BL mice that had lost their delayed-type hypersensitivity to MLM during the course of a disseminated infection showed a fairly strong antibody response to three antigens and a weak response to three others. Since almost the same spectrum of antibodies, although in lower titres, could be demonstrated in C57BL mice with intact delayed-type hypersensitivity to the bacilli, these antibodies did not seem to interfere with the expression of cell-mediated immunity.     

瘤型麻风动物模型的IL-2测定和T细胞的电镜观察

鼠麻风杆菌(Mycobacterium lepraemurium,MLM)感染的BALB/c小鼠是类似瘤型麻风的动物模型。应用这一模型,作者研究了MLM感染小鼠IL-2产生的动态变化。结果表明,MLM感染小鼠脾细胞IL-2的生成明显低于正常小鼠,二者有显著性差异(p<0.01)。为了探索MLM感染的T淋巴细胞的结构与功能的关系,作者对感染动物的T淋巴细胞作了电镜观察。电镜下发现,感染小鼠脾脏的T淋巴细胞浆内有MLM的存在。本文对以上现象进行了讨论。     

Consequences of Smallpox Vaccination in Leprosy Patients

This study illustrates the consequences of smallpox revaccination in 45 lepromatous, 28 tuberculoid, and 47 normal individuals. Results obtained with intradermal inoculations indicated that the patients with leprosy were associated with a relative anergy against the vaccinia virus, the anergy being minimal in the tuberculoid leprosy but marked in the cases with lepromatous leprosy. Major vaccinial reactions were observed more often in patients with lepromatous leprosy than in the controls or patients with tuberculoid leprosy. Furthermore in a patient with lepromatous leprosy, vaccinia necrosum also developed. The smallpox vaccination with live virus also appeared as a provocative factor for the precipitation of lepra reaction in the lepromatous leprosy cases. After 3 weeks of vaccination, the frequency of the specific humoral antibody response was the same in the tuberculoid patients and controls while it was higher in the cases with lepromatous leprosy. The prevaccination titer of total hemagglutination inhibition antibody was significantly higher in the lepromatous leprosy cases. However, the postvaccinial, humoral antibody response of the lepromatous patients was of the same magnitude as that observed in the normal individuals, and it was mainly due to a 2-mercaptoethanol-resistant antibody.     

Risk and protective factors for leprosy development determined by epidemiological surveillance of household contacts

Household contacts of leprosy patients are the group with the highest risk of developing the disease, and although many risk or prevention factors have been identified, they have not been employed in leprosy-monitoring programs. This investigation aimed to establish the relative risks or the preventive effects of the presence of BCG vaccination, the Mitsuda test, and the ML-Flow assay. Household contacts (1,396) were monitored for a 5-year period. Twenty-eight contacts (2%) developed leprosy and had their clinical and operational classifications established. All immunological tests were performed, and intradermal BCG vaccination was given after the BCG scar count. Of the affected contacts, 75% developed the disease in the first year, and 71.4% were classified as having paucibacillary forms. Contacts of lepromatous leprosy patients presented a 3.8-fold-higher risk of developing leprosy. BCG vaccination and the Mitsuda test showed a protective effect against leprosy of 0.27 (at least one scar) and 0.16 (>7 mm), respectively, and the positive ML-Flow test indicated a relative risk approximately sixfold higher for occurrence of the disease. All unfavorable combinations of two and three assays generated significant risk values that ranged from 5.76 to 24.47, with the highest risk given by the combination of no BCG scar, negative Mitsuda test, and positive ML-Flow test. We suggest that the BCG vaccination may be given to stimulate Mitsuda test positivity, reducing the patient's risk of developing multibacillary forms. The high significance of these tests may have a great impact on programs to monitor contacts and should be used to improve early detection and treatment.     

Thymus-dependent lymphocytes in leprosy. II. Effect of chemotherapy on T-lymphocyte subpopulations

The basis of the immunological unresponsiveness seen in leprosy patients is unknown. Untreated lepromatous leprosy patients display an unspecific cellular anergy which disappears with treatment, leaving an anergy specific forMycobacterium leprae. These patients suffer from a complication, erythema nodosum leprosum, characterized by a recurrent eruption of tender skin nodules disappearing in 2 to 3 days. These nodules show a histological picture reminiscent of an Arthus reaction. Erythema nodosum leprosum can occur in untreated patients but it is more frequent in those receiving effective chemotherapy, and this has been thought to be due to massive release of antigens from the bacilli. By using monoclonal antibodies detecting different subpopulations of human peripheral blood T lymphocytes, we have shown that both borderline lepromatous leprosy patients had increased circulating suppressor cells (P<0.001) while the total number of T cells was within the normal range. The suppressor-cell population decreased with the duration of treatment, the change being evident at as early as 21 days. Five patients developed erythema nodosum leprosum during the study period. In all these patients the number of suppressor cells was decreased prior to the complication, increasing to original values with clinical recovery from this syndrome. There was no significant effect on T-lymphocyte subpopulations during chemotherapy of borderline tuberculoid leprosy patients. It seems that antileprosy chemotherapy precipitates erythema nodosum leprosum by interfering with immunoregulatory T cells.     

The role of interleukin-2 (IL-2) in the specific unresponsiveness of lepromatous leprosy to Mycobacterium leprae: studies in vitro and in vivo

The role of IL-2 in the immunological deficiency of lepromatous leprosy patients towards Mycobacterium leprae have been studied further. After initial stimulation with M. leprae + IL-2, lepromatous lymphocytes could be restimulated with M. leprae alone. The specificity of the responses obtained varied. Some patients gave a stronger response to BCG as compared to M. leprae, while in others a stronger response to M. leprae as compared to BCG was obtained. Studies of the composition of lymphocytes in dermal infiltrates subsequent to injection of killed M. leprae revealed that in both tuberculoid and lepromatous patients, early accumulation of cell staining for both IL-2 receptor and IL-2 were seen. However, with time IL-2 receptor and IL-2 staining lymphocytes diminished in lepromatous infiltrates, while these were maintained in tuberculoid lesions.     

Immunological significance of changes in lymph nodes across the leprosy spectrum

Seventy-seven lymph nodes were examined histologically from sixty-two leprosy patients representing the whole range of the disease spectrum from the high resistance form (tuberculoid) to the specific immunity deficiency form (lepromatous). At the lepromatous end of the spectrum paracortical areas were infiltrated with undifferentiated cells of the histiocyte–macrophage series which failed to eliminate mycobacteria. As resistance to infection increased across the leprosy spectrum, histiocytes became more differentiated eventually appearing epithelioid. This was paralleled by increasing numbers of small lymphocytes in the paracortical areas. In the borderline tuberculoid form of the disease an appearance was seen similar to that found in sarcoidosis. In polar tuberculoid leprosy where there is a high degree of cellular immunity, paracortical areas were well developed and populated with lymphocytes and immunoblasts. The immunological significance of these findings are discussed, especially the relation of the changes in morphological appearance of cells of the histiocyte series to the ability of the patient to develop cell-mediated immune reactions.     

HLA Alleles are Genetic Markers for Susceptibility and Resistance towards Leprosy in a Mexican Mestizo Population

Despite the use of multidrug therapy, leprosy remains endemic in some countries. The association of several human leucocyte antigen (HLA) alleles and gene polymorphisms with leprosy has been demonstrated in many populations, but the major immune contributors associated to the spectrum of leprosy have not been defined yet. In this study, genotyping of HLA‐A, ‐B, ‐DR, and ‐DQ alleles was performed in leprosy patients (n = 113) and control subjects (n = 117) from the region with the highest incidence for the disease in México. The odds of developing leprosy and lepromatous subtype were 2.12‐ and 2.74‐fold higher in carriers of HLA‐A*28, and 2.48‐ and 4.14‐fold higher for leprosy and dimorphic subtype in carriers of DQB1*06. Interestingly, DQB1*07 was overrepresented in healthy individuals, compared to patients with leprosy (OR = 0.08) and the lepromatous subtype (OR = 0.06). These results suggest that HLA‐A*28 is a marker for predisposition to leprosy and the lepromatous subtype and DQB1*06 to leprosy and the dimorphic subtype, while DQB1*07 might be a resistance marker in this Mestizo population.     

Adoptively transferred reactivity to M. leprae in nude mice infected with M. leprae.

Reversal reactions are manifestations of delayed hypersensitivity to M. leprae and are thought to be usually accompanied by manifestations of effective cell-mediated immunity (CMI) as measured by bacterial clearing. These experiments were designed to study the induction of reversal reactions in M. leprae-infected, congenitally athymic nude mice using adoptive transfer of CMI. Splenic cell suspensions derived from unimmunized heterozygous nu/+ mice, and those vaccinated with heat-killed M. leprae, viable BCG and a mixture of the two antigens were diluted to contain 10(4), 10(5), 10(6), 10(7) lymphocytes/0.1 ml and infused intravenously into multibacillary nude mice. The production of reversal reactions in leprous nude mice in response to adoptively transferred CMI was studied in a quantitative fashion. Dose responsive induction of reversal reactions, apparent by footpad inflammation and swelling, decreased morphological indices (MI) of the bacteria and mononuclear cell infiltrations, histopathologically, were observed. For nude mice receiving cells primed with 3.9 X 10(5) living BCG alone, the effective dose 50% (ED50) was 1.0 x 10(6) lymphocytes to induce reversal reactions. For those receiving cells primed with 10(7) M. leprae the ED50 was 3.7 x 10(5) lymphocytes. For nude mice receiving cells primed with a mixture consisting of 1/2 the above dose of BCG + 1/2 the above dose of M. leprae, the ED50 was 6.8 x 10(4) lymphocytes.     

Evidence that the Mechanism of Immunological Tolerance (''Central Failure'') Is Operative in the Lack of Host Resistance in Lepromatous Leprosy

The cellular immune defect in lepromatous leprosy has been studied. The following observations have been made: 1) Three lepromatous patients who had been on anti-leprosy treatment for more than 10 years still failed to respond to M. leprae by lymphocyte transformation (mean 0.2%) while they responded strongly to BCG (mean 34.2%) and PPD (mean 56.1%). 2) Lepromatous serum failed to inhibit M. leprae -induced lymphocyte transformation and M. leprae -induced leukocyte migration inhibition. 3) Lepromatous lymphocytes revealed a reduced capacity to attach M. leprae to their surface. The only experimental condition compatible with the observed characteristics would seem to be a state of immunological tolerance to an antigen (or antigens) of M. leprae. The lasting nature of this non-responsive state suggests that it plays a primary role in the pathogenesis of lepromatous leprosy.     

Failure to induce delayed-type hypersensitivity to Mycobacterium leprae in long-term treated lepromatous leprosy patients.

Lepromatous leprosy (LL) patients whose bacillary load has decreased to almost undetectable levels by long-term chemotherapy failed to develop delayed-type hypersensitivity (DTH) to Mycobacterium leprae antigen following immunization with killed armadillo-derived M. leprae. When these LL patients were immunization with killed armadillo-derived M. leprae. When these LL patients were immunized with killed M. leprae in a mixture with live BCG, only DTH to purified protein derivative (PPD) was induced. These results are further evidence that immunological unresponsiveness to the leprosy antigen of patients with lepromatous leprosy is antigen-specific and non-reversible.     

Passive transfer of immunity into leprosy patients by transfusion of lymphocytes and by transfusion of Lawrence''s transfer factor.

About 1,200 million viable lymphocytes from normal but lepromin- and tuberculin-positive human beings were transfused in four patients of lepromatous and one of tuberculoid leprosy three times at monthly intervals. Three patients of lepromatous leprosy suffered from erythema modosum, whereas the other two developed severe reaction whenever put on the smallest dose of dapsone. In one patient of lepromatous leprosy, minimal improvement or none was observed, whereas in the remaining three cases of lepromatous and one of tuberculoid leprosy, clinical, bacteriological, as well as histological improvement occurred. Two of the five patients started to tolerate the dapsone during the period of study. The present study indicates that immunotherapy might have a definite role in the management of the disease especially in cases with erythema nodosum. Lawrence factor, prepared from leucocytes of normal donors, was transfused three times into four lepromatous leprosy patients who were intolerant to anti-leprosy drugs. The donors were healthy but were tuberculin and lepromin (Mitsuda) positive. The clinical, histological, bacteriological (morphological index), and immunological assessments of the patients were performed before and 5 months after starting the immunotherapy. In two patients conversion of Mitsuda reaction occurred, but there was no appreciable improvement in the clinical, histologic, and bacteriologic status of these patients.     

An isoelectric focusing method for the study of the humoral response against the antigen 85 complex of Mycobacterium bovis BCG in the different forms of leprosy.

Isoelectric focusing was used to separate the three components of the antigen 85 complex of Mycobacterium bovis BCG. Antibody responses of leprosy patients against each Mycobacterium bovis BCG. Antibody responses of leprosy patients against each component were quantitated by densitometric analysis of immunoblot assays. The 85A component was recognized by 40% (8/20) of the lepromin positive and negative healthy subjects, by 76% (19/25) of the tuberculoid and by 96% (24/25) of the lepromatous leprosy sera. In contrast, the 85B component was not stained by the control sera, nor by the tuberculoid leprosy sera but by 64% (16/25) of the lepromatous leprosy sera. The results suggest that antigen 85B contains one or several epitopes that are specifically recognized by sera of lepromatous leprosy patients only.     

Hydrogen peroxide and superoxide production by peripheral blood monocytes in leprosy.

Susceptibility to infection with Mycobacterium leprae, the causative organism of leprosy, is the result of a defect in cell-mediated immunity (CMI). The co-operation of macrophages and T lymphocytes is known to be essential for competent CMI response. In this study we have examined peripheral blood monocytes from a range of leprosy patients in an attempt to identify a possible defect in macrophage function. The ability of these cells to produce hydrogen peroxide and superoxide, two bactericidal metabolites of the monocyte/macrophage, has been measured. Monocytes from leprosy patients were found to be capable of producing normal amounts of hydrogen peroxide and superoxide, and no differences in production were found between tuberculoid, lepromatous and control monocytes. These results suggest that macrophages in leprosy are competent, and that probably a T lymphocyte defect contributes to susceptibility to this disease.     

Thymus-Dependent Lymphocytes of Peripheral Blood in Leprosy Patients

Study of the numbers of thymus-derived lymphocytes by the rosette assay (T-RFC) in patients with leprosy reveals that lower than normal numbers of T-RFC are regularly seen in those patients with the active lepromatous form of this disease. Essentially normal numbers of T-RFC were found in inactive lepromatous, borderline, and indeterminate types of leprosy. The lowest percentages and lowest absolute numbers of T-RFC were encountered in patients with lepromatous leprosy resistant to chemotherapy. Patients with lepromatous leprosy complicated by erythema nodosum leprosum show numbers of T-RFC that are more nearly normal than the numbers of T-RFC in patients with uncomplicated lepromatous leprosy. These findings are discussed with respect to the pathogenesis of lepromatous leprosy and the T-RFC deficiency demonstrated in this disease. The possibility that transient defects in T-RFC numbers or function may predispose to lepromatous leprosy is proposed.     

Identification of an immunostimulating protein from Mycobacterium leprae.

Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.