Enoyl-coenzyme A hydratase and antigen 85B of Mycobacterium habana are specifically recognized by antibodies in sera from leprosy patients.

Leprosy is an infectious disease caused by Mycobacterium leprae, which is a noncultivable bacterium. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. M. habana is a cultivable nonpathogenic mycobacterium and candidate vaccine for leprosy, and several antigens that cross-react between M. leprae and M. habana have been discovered. The aim of the present study was to extend the identification of cross-reactive antigens by identifying M. habana proteins that reacted by immunoblotting with antibodies in serum samples from leprosy patients but not with antibodies in sera from tuberculosis (TB) patients or healthy donors (HDs). A 28-kDa antigen that specifically reacted with sera from leprosy patients was identified. To further characterize this antigen, protein spots were aligned in two-dimensional polyacrylamide gels and Western blots. Spots cut out from the gels were then analyzed by mass spectrometry. Two proteins were identified: enoyl-coenzyme A hydratase (lipid metabolism; ML2498) and antigen 85B (Ag85B; mycolyltransferase; ML2028). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of lepromatous leprosy, which is the most frequent form in Mexico.

Western blot profile in HIV infection.

BACKGROUND: Although the overall sensitivity and specificity of the western blot (WB) test for detection of antibodies to various viral proteins is high, there has been a substantial difference in the timing of the appearance of antibody bands and their intensities during different stages of HIV infection. AIMS: Mapping different band patterns of Western blot results and correlating them with stages of HIV infection. METHODS: We performed a retrospective study with 1,467 HIV-1 infected cases confirmed by WB test between January 2002 to July 2005, with the objective of mapping different band patterns of western blot results and determining whether the presence or absence of certain bands was associated with any specific stage of HIV infection. For the interpretation of the WB results in this study, the guidelines recommended by NACO, India were followed. RESULTS: Reactivity with all the bands was the most commonly observed WB pattern, occurring in 92.91% (1363/1467) of cases, whereas the other 7.09% showed uncommon band patterns. Of all individual bands, p31 band was the most frequently missing one, absent in 7.09% cases. On classifying the WB reactive cases by the WHO clinical staging system, 38.45% (564/1467) were in Stage 1, 47.99% (704/1467) in stages 2 and 3 and 13.56% in stage 4. Correlation of CD4 cell counts with the various uncommon band patterns showed that only 5.56% (4/72) had counts in the 200-500 cells/microl range, whereas 45.83% and 48.61% had counts of < 200 and> 500 cells/microl respectively. CONCLUSION: Interpretation of the WB band pattern in combination with clinical features may be occasionally useful in predicting the stage of HIV infection.

ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy.

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.

[Regulation of the immune response: the TH1/TH2 concept]. / Regulation der Immunantwort: das TH1-/TH2-Konzept.

The immune system has different possible ways of reacting to an antigen. The choice of an appropriate immune response is determined by the manner of antigen presentation, the amount of antigen, the localization of antigen uptake, the type of antigen presenting cell, the genetic predisposition of the individual and the presence of certain cytokines released by antigen presenting or other inflammatory cells. An immune response which is not not appropriate can lead to clinical symptoms or insufficient clearance of an infectious agent. This is well-illustrated in the example of lepra lepromatosa (insufficient, since humoral immune response to an intracellular agent) or lepra tuberculosa (complete clearance of Mycobacterium leprae). A decisive step for the type of immune response is the stimulation of different T-cell subpopulations. CD4 or CD8 T-cells can be further subdivided by a distinct cytokine production. So-called TH1 cells predominantly produce cytokines, which stimulate a cellular immune response (IFN gamma, IL-12, IL-2). In contrast, TH2 cells predominantly produce IL-4 and IL-5. These cytokines boost an IgE-mediated allergic reaction and inflammation. Although the TH1/ TH2 distinction is frequently not absolute, as overlaps can frequently be observed, this classification is useful for better understanding of immune reactions in various diseases. Moreover, since TH1- and TH2-related cytokines act antagonistically, therapeutic strategies are under development which strengthen e.g. a TH2 immune response in TH1 dominated diseases and vice versa.

Recognition of Mycobacterium leprae recombinant 18,000 MW epitopes by IgG subclasses in leprosy.

IgG subclasses are known to be differentially regulated by cytokines (elaborated by activated T cells), which act as growth factors and immunoglobulin switch factors on B cells. In leprosy, we have previously shown that IgG subclass antibodies to a purified recombinant antigen of Mycobacterium leprae (18,000 MW) are restricted to IgG1 and IgG3 across the disease spectrum. The only significant difference observed was that lepromatous patients with low to undetectable T-cell responses showed a strong correlation between IgG1 and IgG3 (P < 0.001) antibodies while tuberculoid patients who showed strong T-cell responses did not show such a correlation. To examine if these differences were related to T-cell-mediated class switching in tuberculoid leprosy patients, we have studied epitope recognition by IgG1 and IgG3 using a panel of synthetic peptides spanning the 18,000 MW molecule in an enzyme-linked immunosorbent assay (ELISA). In lepromatous patients there was little similarity in peptide recognition by IgG1 and IgG3, with IgG1 recognition being restricted to a single dominant carboxy-terminal peptide while the IgG3 antibodies recognized a diverse set of peptides in the N-terminal half of the 18,000 MW molecule. In tuberculoid patients both IgG1 and IgG3 antibody showed recognition of similar peptides in the N-terminal half of the 18,000 MW molecule. Our results therefore support the hypothesis that immunoglobulin class switching is occurring in tuberculoid but not in lepromatous patients.

Expressao do antigeno de 18kDa de Mycobacterium leorae na levedura Saccaromyces cerevisisae e estudo das propriedades imunogenicas do antigeno recombinante / Expression of the antigen of 18kDa of Mycobacterium leprae na levedura Saccharomyces cerevisiae and study of imonogenics properties of recombinant antigen

Dois diferentes sistemas de expressao do antigeno de 18 kDa de M. leprae (p18) em S. cerevisiae, um intracelular e outro de secrecao, foram desenvolvidos. Ambos os sistemas mostraram-se efetivos para a expressao, mas a purificacao da p18 secretada mostrou-se mais simples. Comparando diferentes cepas hospedeiras e condicoes de cultivo, foi obtido um sistema de secrecao de alto rendimento (mais de 100 mg de proteina biologicamente ativa por litro). A p18 foi purificada do meio de cultura da levedura por precipitacao, seguida de cromatografias de troca ionica e por filtracao em gel. As propriedades imunologicas da proteina recombinante, nativa ou previamente irradiada com raios 'GAMA' foram analisadas em camundongos. Ambas as preparacoes desencadearam producao de anticorpos e reacao de hipersensibilidade tardia, correspondentes as respostas humoral e celular, respectivamente. Em adicao, a irradiacao previa do antigeno potencializou sua imunogenicidade a nivel celular. Estes resultados demonstram ser esta proteina forte candidata para utilizacao em novos testes cutaneos para a monitorizacao da resposta celular contra M. leprae

Evaluation of diversified antigens for detection of Mycobacterium leprae antibodies from leprosy patients and contacts.

A comparative evaluation of diversified antigens in ELISA has been made for detection of M. leprae antibodies in the sera of leprosy patients and their contacts. Out of the four antigens, namely M. leprae sonicate (ML), phenolic glycolipid I (PGL-1), M. habana sonicate (MH) and its arabinomannan (AM), the cross reactive antigens (MH,AM) have comparatively detected more number of leprosy cases. Homologous antigens (ML, PGL-1) have lower detection level. Use of MH and AM for detection of mycobacterial antibodies have been discussed and advocated for epidemiological studies of leprosy/tuberculosis.

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.

Recognition of Mycobacterium leprae antigens with antibodies present in sera from patients with lepromatous leprosy.

A great diversity of antigens from Mycobacterium leprae have been described. One practical approach should be to utilize them as markers to indicate when a household contact is at risk of becoming infected and then moving to an active form of leprosy. For this purpose, sonic extracts of M. leprae were fractionated in 10% SDS-PAGE under reducing conditions. The fractionated proteins were then transferred to nitrocellulose sheets and incubated with sera from lepromatous leprosy cases, their contacts, and normal subjects in order to reveal the frequency of antigen recognition of each set of sera. The results showed that sera from lepromatous leprosy patients frequently recognized two proteins, one of approximately 28 kDa and the other of approximately 65 kDa, when compared with the sera from normal subjects. The contacts frequently recognized an approximately 16-kDa antigenic band, while sera from normal subjects recognized one protein of approximately 18 kDa. According to the results, the four recognized proteins from M. leprae can be considered markers of the above conditions (approximately 65 kDa, approximately 28 kDa for lepromatous leprosy, approximately 16 kDa for contacts, and approximately 19 kDa for normal subjects). From these, an easy serological test, such as an ELISA, can be developed to predict if a contact is moving toward lepromatous leprosy before detection of the actual clinical signs or symptoms.

Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning.

The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC16); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.

Detection of antibodies to human nerve antigens in sera from leprosy patients by ELISA.

Anti-neural antibodies have been implicated to play a role in the pathogenesis of nerve damage in leprosy patients. To find the relationship between anti-neural antibodies and clinical findings, we attempted to detect antibodies against neurofilament-enriched proteins by ELISA in sera from leprosy patients. Of 289 sera from leprosy patients, 74 (25.6%) had significant anti-neural antibodies; in contrast, 1 (5.0%) of 20 tuberculosis patients and 11 (7.1%) of 154 controls were seroreactive to nerve antigen. When clinical types were considered, a significant level of anti-neural IgG antibodies was detectable in 53 (30.1%) of 176 sera from lepromatous patients compared with 21 (18.6%) of 113 sera from tuberculoid patients, indicating that lepromatous patients were more likely to be seropositive to nerve antigens in ELISA. Some of the ELISA-reactive sera showed antibody reactivity with 38-kD, 40-kD and 43-kD nerve antigens in Western blotting analysis. There was no apparent correlation between seroreactivity to nerve antigens and bacterial load in leprosy patients. Although there was no statistical significance, anti-neural antibodies were detectable more often among the patients on chemotherapy than the untreated and among the patients with erythema nodosum leprosum than without. The results, therefore, suggest that anti-neural antibodies are elicited during the course of leprosy and may be associated with the extensiveness of nerve involvement in the patients.

Características epidemiológicas da hanseníase indeterminada, casos detectados no Estado de Säo Paulo no período de 1959 a 1973 e de 1977 a 1984 / Epidemiological characteristics of indeterminate leprosy: detected case in Säo Paulo State during the years 1959/1973 and 1977/1984

Descreve-se e avalia-se as características epidemiológicas da Hanseníase Indeterminada no Estado de Säo Paulo em dois períodos (1959 a 1973 e de 1977 a 1984) de uma amostra de casos levantados a partir de laudos histopatológicos e de prontuários clínicos da Divisäo de Hansenologia e Dermatologia Sanitária do Instituto de Saúde da Secretaria de Saúde do Estado de Säo Paulo. Estes períodos correspondem a diferentes estratégias políticas de Saúde, sendo que no primeiro, que denominamos vertical, a política de atençäo à endemia era centralizada e dispensarial e, no segundo, descentralizada e acoplada à atençäo primária de saúde da rede de unidades básicas de saúde do Estado. As variáveis analisadas säo descritas e comparadas entre os dois períodos

Polyspecificity of human monoclonal antibodies reactive with Mycobacterium leprae, mitochondria, ssDNA, cytoskeletal proteins, and the acetylcholine receptor.

The origin of autoantibodies against ubiquitous autoantigens (e.g., single-stranded (SS) DNA, cytoskeletal proteins, mitochondria) is obscure. Patients with lepromatous leprosy have many such autoantibodies in their serum. In order to study the polyspecificities of human autoantibodies expressed during infection with Mycobacterium leprae we prepared human monoclonal antibodies derived from the fusion of peripheral blood lymphocytes of a patient with lepromatous leprosy to the human lymphoblastoid line GM 4672. Hybridomas were tested for binding to a DNAse-treated sonicate of M. leprae and a panel of autoantigens. Of the primary (uncloned) cultures, 14% bound ssDNA, 35% bound M. leprae, 11% bound both M. leprae and ssDNA, and 16% bound to mitochondria. Several also bound to the acetylcholine receptor of Torpedo marmorata. Monoclonal antibodies derived from separate primary cultures revealed similar cross-reactions between several autoantigens and M. leprae. In addition, one antibody was identified which bound to mitochondria and the acetylcholine receptor, and which was recognized by an anti-idiotypic antibody which bears the "internal image" of the acetylcholine receptor. These results suggest that antigenic mimicry may play a role in eliciting autoantibody expression from the immune repertoire.

Enzyme immunoassay for IgG rheumatoid factor combining with homologous IgG.

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')2 of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%-85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.

Use of recombinant antigens expressed in Escherichia coli K-12 to map B-cell and T-cell epitopes on the immunodominant 65-kilodalton protein of Mycobacterium bovis BCG.

In gene libraries of Mycobacterium bovis BCG, Mycobacterium tuberculosis, and Mycobacterium leprae, recombinants were frequently encountered that expressed an immunodominant 65-kilodalton (kDa) protein antigen that was shown to react with a high proportion of mycobacterium-reactive human and murine T cells and murine monoclonal antibodies. In this study, recombinant antigens were used to map T-cell and B-cell epitopes on the M. bovis BCG 65-kDa protein that was previously designated MbaA. Four different T-cell-epitope-containing regions (amino acid residues 1 through 16, 17 through 61, 85 through 108, and 235 through 279) were defined that were recognized by seven T-cell clones from patients with tuberculoid leprosy. These regions are distinct from two previously described T-cell epitopes recognized by T cells from a tuberculosis patient. As T-cell clones restricted by different class II determinants were shown to be specific for different regions on the 65-kDa protein, the presented data suggested that the products of different human leukocyte antigen class II loci and alleles present different parts of MbaA to the immune system. B-cell epitopes recognized by 20 monoclonal antibodies were assigned to eight different regions of MbaA. Using 15 of these antibodies, we previously showed that MbaA was antigenically related to a common antigen present in many bacterial species. The dispersed localization of the involved epitopes defined here shows that various different parts of MbaA are indeed conserved. These results show that well-defined recombinant antigens are useful tools for the localization of both B- and T-cell-epitope-containing regions of a protein. Peptides synthesized from the sequences of such regions may then exactly define the epitopes relevant for the development of specific diagnostic tests or of vaccines against mycobacteria.

T cell epitopes on the 36K and 65K Mycobacterium leprae antigens defined by human T cell clones.

To identify the molecular localization and specificity of Mycobacterium leprae antigenic determinants inducing T cell activation, we studied the reactivity of M. leprae-reactive T cell clones from two tuberculoid leprosy patients towards a battery of different mycobacterial strains and purified mycobacterial antigens. Of the 38 T cell clones tested 8 appeared to be M. leprae specific (specificity A), another 8 were cross-reactive with at least one of the three mycobacterial strains, M. lepraemurium, M. vaccae and M. scrofulaceum (specificity B), 5 were reactive with most but not all strains (specificity C) and the remaining 18 were reactive with all 17 mycobacterial strains tested (specificity D), but not with nonmycobacterial antigens. All T cell clones were tested with the 36K and 65K antigen isolated from M. leprae, and with the M. leprae and M. bovis BCG 65K proteins produced in E. coli by recombinant DNA. Four T cell clones appeared to recognize epitopes on the 36K antigen, nine T cell clones recognized the 65K antigen. These 2 M. leprae antigens, 36K and 65K, thus seem to contain major T cell epitopes. At least 3 different epitopes could be defined on the 36K antigen of which one is M. leprae specific, one of specificity B and one of specificity C. Two distinct epitopes were discerned on the 65K antigen of which one is M. leprae specific and one of specificity D. The M. leprae-specific epitopes on the 36K and 65K antigen may help in the development of a specific serodiagnostic and skin test.