Specific IgG antibody responses may be used to monitor leprosy treatment efficacy and as recurrence prognostic markers.

Although curable, leprosy requires better diagnostic and prognostic tools to accompany therapeutic strategies. We evaluated the serum samples of leprosy patients from Venezuela and Brazil for reactivity against the specific recombinant proteins, ML0405 and ML2331, and the LID-1 fusion protein that incorporates both of these antigens. Antigen-specific IgG was highest in lepromatous leprosy patients (LL) and decreased across the disease spectrum, such that only a small subset of true tuberculoid patients (TT) tested positive. The impact of multidrug therapy (MDT) on these antibody responses was also examined. Several years after treatment, the vast majority of Venezuelan patients did not possess circulating anti-LID-1, anti-ML0405, and anti-ML2331 IgG, and the seropositivity of the remaining cases could be attributed to irregular treatment. At discharge, the magnitude and proportion of positive responses of Brazilian patients against the proteins and phenolic glycolipid (PGL)-I were lower for most of the clinical forms. The monthly examination of IgG levels in LL patient sera after MDT initiation indicated that these responses are significantly reduced during treatment. Thus, responses against these antigens positively correlate with bacillary load, clinical forms, and operational classification at diagnosis. Our data indicate that these responses could be employed as an auxiliary tool for the assessment of treatment efficacy and disease relapse.

Inorganic particles in the skin of inhabitants of volcanic areas of Central America: their possible immunomodulatory influence in leishmaniasis and leprosy.

We have evaluated biopsies from patients with atypical nodular and typical ulcerated lesions of cutaneous leishmaniasis, from leishmanin reactions and skin from normal individuals from Nicaragua, Honduras and Guatemala for the presence of inorganic particles using confocal microscopy with a polarised light source and conventional histopathological techniques. Analysis by semiquantitative confocal microscopy permitted the demonstration of significantly larger numbers of particles in atypical lesions. Silica and aluminium, important components of these particles, were less abundant in particles from normal skin. The histology of these atypical lesions, characterised by 'naked' sarcoidal granulomas with epithelioid differentiation but very few lymphocytes, was very similar to the histological reaction observed after 14 days in persisting inflammation at leishmanin skin test sites. The presence of these unusual lesions in areas of Central American countries characterised by the presence of large amounts of volcanic ash, as well the unexpectedly low prevalence of leprosy in Central America, suggest that environmental factors may contribute significantly to the frequency and clinical manifestations of these infections. Among possible environmental features, the presence of inorganic particles with immunomodulatory properties in the skin may be a significant factor.

Lepra: evaluación inmunonológica de contactos intradomiciliarios / Leprosy: an assessment of contacts inmunonológica intradomiciliary

Los contactos intradomiciliarios de pacientes con lepra representan una población con riesgo de infección . El uso combinado de pruebas cutáneas y ELISA revela el grado de sensibilización, la capacidad de respuesta inmunológica y casos subclínicos de la enfermedad. Con base en lo anteriormente expuesto y con la finalidad de justificar el uso de estas pruebas, de rutina en otros contactos, se entrevistó el evaluó clínicamente a 211 contactos intradomiciliarios, de 32 casos de lepra registrados. Se colocó a los contactos lepramina intradérmica y se determinó niveles de anticuerpos específicos contra M. leprae (prueba de ELISA con PGL-1). De la población evaluada, 99.88 por ciento presentó reacción de Fernández negativa y 2/3 de ella presentó una reacción de Mitsuda positiva. Sólo 30.85 por ciento constituyó un grupo de riesgo por presentar reacción de Mitsuda negativa. Al correlacionar las pruebas cutáneas con el ELISA se mostró que ninguno presentaba lepra en fase subclínica y que un sólo caso de ELISA débilmente positivo resultó ser una infección pasada autolimitada. No se justifica usar todas las pruebas inmunológicas, en todos los contactos. Se recomienda usar pruebas cutáneas para detectar grupos de riesgo y para orientar quimiprofilaxis, reservado el uso del ELISA sólo para grupos de riesgo demostrado.

Control de la lepra en Venezuela después de más de cinco décadas de desarrolo

La lucha antileprosa se incia en Venezuela en el siglo XIX, y se refuerza a comienzos del siglo XX. Con la creación del Ministerio de Sanidad en 1936 se inicia el servicio de lucha antileprosa y en 1946 se crea la División de Lepra, que se convertirá después en el Departamento de Dermatología, el cual actualmente forma parte del Instituto de Biomedicina, que es organismo rector de esta actividad a nivel estatal, y cuyas actividades a nivel operativo se realizan a través de los 31 servicios regionales de Dermatología Sanitaria. En 1985 se inició la aplicación de la poliquimioterapia supervisada como tratamiento de elección. La evolución de la lepra en Venezuela desde 1946 se caracteriza por un aumento de la detección y la prevalencia en los años inmediatos a esta fecha, con un descenso posterior desde la década del 60, estabilizándose la deteción desde inicios de la década del 80, con tasas alrededor de 0'25 (algo más de 500 casos) por 10.000 habitantes por año. Para este período de ha mantenido el descenso de la prevalencia, la cual mostró cambios bruscos en 1982 y 1995, por actuatualización de los registros. En nivel de eliminación de la lepra como problema de salud (de acuerdo al criterio de la OMS de una tasa de prevalencia inferior a uno ano por 10.000 habitantes), se alcanzó en Venezuela en 1997. En las entidadades federales, soleamente cuatro de las 23 (Apure, Barinas, Dojedes y Portuguesa) mantienen tasas de prevalencia por encima del nivel de eliminación. Dado el hecho de haber alcanzado el nivel de eliminación, manteniéndose relaticamente estables el número de casos nuevos y la tasa de detección, se propone que el criterio para clasificar a un país como "en fase de eliminación) sea más estricto e incluya, además de la tasa de prevaelncia, datos relativos a la detección de casos. El tipo clínico predominante es el multibacilar. Las tasas de detección por edade muestran un aumento gradual al avanzar en edad. La racón masculino/femenino se mantiene alrededor de 2. Un 10% de los casos presentaron algún grado de discapacidad, aunque discreta en la mayoría de los casos. Existe un porcentaje de casos nacidos en el ixterior que es superior a la proporción de población con esta característica en el país. La mayor proporción de casos proviene del área urbana, especialmente de las zonas marginales, aun cuando las tasas son mayores para la zona...

Serologic response to mycobacterial proteins in hansen's patients during multidrug treatment.

Humoral immune responses were studied in 24 leprosy patients treated with multidrug therapy (MDT) and 16 contacts. The patients were monitored for 2 to 3 years with repeated determination of IgG antibody levels directed to different mycobacterial proteins (Mycobacterium tuberculosis, Mt70; M. bovis, Mb65; M. leprae, Ml36, 28, 18, 10 kDa, and the complete protein M. leprae extract, MLSA). All recombinant antigens were used at 5 micrograms/ml concentration and the complete soluble M. leprae extract at 2 micrograms/ml. The results shown in this study reveal a clear decline in IgG antibodies directed toward mycobacterial proteins in the 12 multibacillary (MB) patients when they were submitted to MDT. Initially we found strong reactivity toward complete cytosolic protein and M. leprae membrane protein. The most reactive recombinant proteins in MB patients were Ml10, Ml36, Mt70 kDa and, finally, Ml18 kDa when compared to the paucibacillary (PB) group. After treatment was completed all lepromatous and borderline lepromatous patients showed low or undetectable levels as compared with their initial values before starting treatment.

A follow-up study of multibacillary Hansen's disease patients treated with multidrug therapy (MDT) or MDT + immunotherapy (IMT).

Multibacillary (MB) leprosy patients treated with multidrug therapy (MDT) or MDT + immunotherapy (IMT) with BCG + heat-killed Mycobacterium leprae were tested annually for their ability to proliferate in vitro to the mycobacterial antigens BCG, M. leprae soluble extract, and intact M. leprae. IgM antibody responses to phenolic glycolipid I (PGL-I) were measured, as well as serum nitrite levels in patients' sera, before, during and after treatment. Patients who received only MDT did not present cellular reactivity to intact M. leprae antigens, in contrast to the results obtained with BCG, which elicited reactivity at time zero, that increased after treatment. Regarding PGL-I antibody variations in relation to the initial value, we observed a statistically significant marked decrease at the end of 2 years which continued to fall in successive evaluations. MB patients showed high initial serum nitrite concentrations which dropped drastically with treatment. This decay was apparently associated with the bacillary load present in these patients. The group submitted to IMT + MDT showed high and long-lasting T-cell responses to mycobacterial antigens in a significant number of initially unresponsive MB patients. There was a marked increase to M. leprae soluble extract and BCG, as well as a more variable response to whole bacilli. The antibody levels in this group of patients are sustained for a somewhat longer period and decreased more slowly during the 5-year follow up.

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.

A longitudinal study of immunologic reactivity in leprosy patients treated with immunotherapy.

More than 150 leprosy patients treated with multidrug therapy (MDT) plus immunotherapy (IMT) with a mixture of heat-killed Mycobacterium leprae plus live BCG were studied in relation to humoral and cell-mediated immune responses. Many previously had received prolonged sulfone monotherapy. Patients received 2 to 10 doses of IMT in a period of 1 to 3 years, depending upon their clinical form of leprosy. The patients were followed up for 5 to 10 years with repeated determinations of antibody levels to phenolic glycolipid-I; lymphoproliferative (LTT) responses to soluble extract of M. leprae, to whole bacilli and to BCG, skin-test responses and bacterial indexes (BIs). After MDT plus IMT there was a statistically significant decrease of antibody levels in the multibacillary (MB) group. The BI decreased proportionally to the ELISA results. LTT increased to M. leprae antigens, especially to soluble extract, in a high percentage of these patients (34% of LL patients positive). Lepromin positivity in MB patients increased from 5% initially positive to 75% at the cut-off during this follow up. These results show substantial early and persistent cell-mediated reactivity to M. leprae in many MB patients treated with MDT-IMT, confirming and expanding previously published data.

Immune response in healthy volunteers vaccinated with BCG plus killed leishmanial promastigotes: antibody responses to mycobacterial and leishmanial antigens.

Antibody (IgG) responses to mycobacterial (BCG; PPD; Mycobacterium leprae soluble antigen, MLSA) and leishmanial (Leishmania mexicana LV4) antigens were measured in 208 initially PPD and leishmanin skin-test negative volunteers divided into four vaccine groups as follows: 68 received BCG plus killed promastigotes (group A), 47 received BCG alone (group B), 47 received killed promastigotes alone (group C), and 46 formed the diluent control (placebo, group D). Three vaccine doses were administered at 8-12 week intervals. Vaccinees were bled immediately prior to each vaccination, and again at 3- and 12-month follow-up. Skin tests were performed prevaccination, and again at the 3- and 12-month follow-up. Anti-BCG, anti-PPD and anti-MLSA IgG levels increased significantly in groups A and B receiving BCG. The presence of leishmanial antigen (with BCG) in the inoculum suppressed the IgG response to Mycobacterium tuberculosis/Mycobacterium bovis-related (PPD and BCG), but not M. leprae-related (MLSA)-related, antigens. A small but significant increase (relative to prevaccination level) in response to MLSA, but not to BCG or PPD was observed in the non-BCG-vaccinated groups. The background level of response to mycobacterial and leishmanial antigens was higher in the Venezuelan vaccinees than in non-endemic (British) volunteers. Responses to leishmanial antigen were not enhanced in the two vaccine groups receiving killed promastigotes (with/without BCG) compared with the BCG alone and placebo groups. Instead, all vaccine groups showed a pattern of response consistent with either (i) a response to the skin-test antigen or, more likely, (ii) seasonal endemic exposure to leishmanial antigen. Interestingly, this endemic response to leishmanial antigen was enhanced in the vaccine groups receiving BCG, despite the fact that group B received no leishmanial antigen in the vaccine inoculum. When prevaccination IgG levels (mean + 3 standard deviations) were used to determine a negative cut-off, a low percentage (< 38%) of vaccinees converted to responder status for either anti-mycobacterial or anti-leishmanial responses, and those who did would be classified as 'low-responder' status compared with titres observed in severe forms of disease. Hence, although there was evidence for a background endemic response to both leishmanial and mycobacterial antigens, there was no evidence that vaccination per se led to a potentially disease exacerbatory level of TH2-associated antibody response especially with respect to the anti-leishmanial response.(ABSTRACT TRUNCATED AT 400 WORDS)

BCG vaccination protects against leprosy in Venezuela: a case-control study.

A total of 64,570 household and other close contacts of about 2000 leprosy cases were screened for eligibility for entry into a trial of a new leprosy vaccine. The screening procedure included a clinical examination for leprosy and for the presence of BCG and lepromin scars. Ninety-five new cases of leprosy were identified, and the prevalence of BCG and lepromin scars among them was compared with similar data from matched controls selected from among those with no evidence of leprosy. The difference in the prevalence of BCG scars in the two groups was used to estimate the protection against leprosy conferred by BCG vaccination. One or more BCG scars was associated with a protective efficacy of 56% (95% confidence limits 27% to 74%). There was a trend of increasing protection with four or more BCG scars, but this was not statistically significant. There was no evidence that the efficacy of BCG varied with age or according to whether or not the contact lived in the same household as a case. The protective effect was significantly higher among males, and was significantly greater for multibacillary than for paucibacillary leprosy.

Preliminary study of cellular immunity to Mycobacterium leprae protein in contacts and leprosy patients.

Because of the good results obtained in the mononuclear cell (T lymphocyte) proliferative response in tuberculoid leprosy patients and family contacts and healthy Mitsuda-positive volunteers using Mycobacterium leprae soluble extract, we prepared different protein fractions from the soluble extract. We used the T-cell Western blot technique with separation by electrophoresis in SDS-polyacrylamide gels and transfer onto nitrocellulose membranes. Each unstained blot was converted into 18 fractions of antigen-bearing particles and tested with peripheral blood mononuclear cells from 21 individuals including Mitsuda-positive contacts, vaccinated lepromatous leprosy (LL) patients, borderline tuberculoid (BT) patients, and unvaccinated lepromatous patients. The stimulation index (SI) of the contacts was higher to the different fractions in comparison with the leprosy patients. They showed four peaks of stimulation to fractions 66-55, 45-29, 22-18, and 14 kDa. The second highest responders were BT patients, followed by vaccinated LL patients. The unvaccinated patients did not respond significantly to any of the fractions (SI less than 1).

Immunoprophylactic trial with combined Mycobacterium leprae/BCG vaccine against leprosy: preliminary results.

In an attempt to find a vaccine that gives greater and more consistent protection against leprosy than BCG vaccine, we compared BCG with and without killed Mycobacterium leprae in Venezuela. Close contacts of prevalent leprosy cases were selected as the trial population since they are at greatest risk of leprosy. Since 1983, 29,113 contacts have been randomly allocated vaccination with BCG alone or BCG plus 6 x 10(8) irradiated, autoclaved M leprae purified from the tissues of infected armadillos. We excluded contacts with signs of leprosy at screening and a proportion of those whose skin-test responses to M leprae soluble antigen (MLSA) were 10 mm or more (positive reactions). By July, 1991, 59 postvaccination cases of leprosy had been confirmed in 150,026 person-years of follow-up through annual clinical examinations of the trial population (31 BCG, 28 BCG/M leprae). In the subgroup for which we thought an effect of vaccination was most likely (onset more than a year after vaccination, negative MLSA skin-test response before vaccination), leprosy developed in 11 BCG recipients and 9 BCG/M leprae recipients; there were 18% fewer cases (upper 95% confidence limit [CL] 70%) in the BCG/M leprae than in the BCG alone group. For all cases with onset more than a year after vaccination irrespective of MLSA reaction the relative efficacy was 0% (upper 95% CL 54%; 15 cases in each vaccine group). Retrospective analysis of data on the number of BCG scars found on each contact screened suggested that BCG alone confers substantial protection against leprosy (vaccine efficacy 56%, 95% CL 27-74%) and there was a suggestion that several doses of BCG offered additional protection. There is no evidence in the first 5 years of follow-up of this trial that BCG plus M leprae offers substantially better protection against leprosy than does BCG alone, but the confidence interval on the relative efficacy estimate is wide.

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones.

Functional subsets of human T cells were delineated by analyzing patterns of lymphokines produced by clones from individuals with leprosy and by T cell clones of known function. CD4 clones from individuals with strong cell-mediated immunity produced predominantly interferon-gamma, whereas those clones that enhanced antibody formation produced interleukin-4. CD8 cytotoxic T cells secreted interferon-gamma. Interleukin-4 was produced by CD8 T suppressor clones from immunologically unresponsive individuals with leprosy and was found to be necessary for suppression in vitro. Both the classic reciprocal relation between antibody formation and cell-mediated immunity and resistance or susceptibility to certain infections may be explained by T cell subsets differing in patterns of lymphokine production.