Abstracts of Congress for Papers and Posters
PM & BM 10
DETECTION OF mRNA CODING FOR PROTEASE ENZYMES IN Mycobacterium leprae ISOLATED FROM HUMAN BIOPSIES
Ribeiro, M.L. 1,2, Tempone, A.J.2, Nery. J.A.2., Albuquerque. E.C.A2, Sarno. E.N.2, Brennan, P.J.3 and Pessolani, M.C.V.2
1Dept. of Medical Biochemistry, ICB, Federal University of Rio de Janeiro 2Leprosy Laboratory. Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil; 3Dept. of Microbiology, Colorado State University, Fort Collins, CO, U.S.A.
The knowledge about the mechanisms of Mycobacterium leprae pathogenesis and the virulence genes responsible for it is very limit. Bacterial proteases have been proposed as virulence factors in a variety of diseases, contributing in different ways to the establishment and maintenance of microorganisms in the host. In this work, we have investigated the in vivo expression by M. leprae of genes annotated as putative proteases in the genome of this pathogen (Cole et al.. Nature 409: 1007-1001, 2001). Five out of 32 protease genes were initially selected for this study: ML0041, ML0176. ML2659. gcp gene (ML0379) and clpC gene (ML0235). These genes code for putative secreted proteases, or for proteases with homology to virulence factors of other microorganisms. M. leprae was purified from biopsies of lepromatous leprosy patients and total bacterial RNA was isolated by guanidine thioeyanate extraction. cDNA was synthesized in a reverse transcriptase reaction with random hexanucleotides. PCR reactions were conducted in the presence of protease specific primers designed for the amplification of the full lengh of the genes. Preliminary results indicate that M. leprae expresses the ML2659 gene, which shares homology with the serine protease pepA, a virulence factor of Pseutlomotias aeruginosa. Additional experiments are under way to further characterize the expression of these genes and to investigate their protease activities.
Supported by CAPES. CNPq and NIAID.NIH.
PM & BM 11
DICHOTOMY OF -238 AND -308 SINGLE NUCLEOTIDE POLYMORPHISMS IN TNF-α GENE: CLINICAL AND BACTERIOLOGICAL EVALUATION IN LEPROSY
P. R. Vanderborghl, M. O. Moraes, J. H. Matos, A. M. Salles, S. E. G. Vasconcellos, V. F. Silva-Filho, T. W. J. Huizinga, T. H. M. Ottenhoff, E. P. Sampaio, E. N. Sarno, A. R. Santos
Tumor necrosis factor alpha (TNF-α) plays a key role in orchestrating the complex events involved in inllanunalion and immune response. The presence of single nucleotide polymorphisms (SNPs) within the promoter region of the TNF-α gene has been associated to a number of diseases. Since the genetic predisposition could be considered as one of the factors in the outcome of different clinical forms of leprosy, the aim of this paper was to investigate the occurrence of (G/A) polymorphisms at positions -238 and -308 within the TNF-α promoter and its possible association with degree of severity. By definition, multi-bacillary (MB) forms was considered severe and the paucibacillary (PB) form. mild. Besides, the bacteriological index (BI) was evaluated among genotyped MB patients in order to investigate the possible influence of each polymorphism on [he levels of bacterial load. The results of this study, which included a total of 631 leprosy patients (MB=401. PB=230) suggest that the -238A allele was associated to the more severe clinical form of leprosy (MB), whereas, the -308A allele with the mild form (PB). These data are in compliance with the BI analyses of MB patients in thai the bacterial load among the -308 carriers was lower while among -238 carriers it was increased.
PM & BM 12
DRUG RESISTANT MycobacleriumlLeprae FROM RELAPSE OR INTRACTABLE LEPROSY CASE
Masanori Matsuoka'. Yoshiko Kashiwabara1. Motoaki Ozaki2 and Sinji Maeda'
'Leprosy Research Center, National Institute of Infectious Diseases. Tokyo, Japan,
2Hyogo Prefeelural Amagasaki Hospital, Hyogo. Japan,
3Osaka City University Graduate School of Medicine. Osaka. Japan
Current strategy against leprosy is mainly based on mulli drug treatment (MDT). On the other hand, some cases of the drug resistant Mycobacterium leprae were reported by deferent study groups. Recently, there have been advances in the elucidation of molecular events responsible for drug resistance in Mycobacteria. Molecular analysis technique lakes place in vivo drug susceptibility test and enables to know correctly the distribution of resistant strains by examining many samples. In this study. The DNA sequences of particular regions of M. leprae, which are responsible for resistance to daposon, rifampin, and fluoroquinolones were analyzed respectively. Samples are collected from Japanese relapsed or intractable cases, newly registered cases in Philippines and Indonesia. For the Japanese cases, 13 out of 16 samples analyzed folP gene, 9 out of 16 analyzed rpoB, 4 out of 8 analyzed gyrA indicated mutation at the position responsible for drug resistant. Results of the samples form Philippines were as follows, folP: 3 mutated/27 examined, ropB: 6 mutated/23 examined. Indonesian samples revealed as fol lows, folP: 2 mutated/27 examined, ropB: 5 mutated/23 examined. Two cases of Philippines regarded resistant daposon and rifampin. Frequent drug resistant cases in Japanese cases may attribute to irregular and/or monotherapy. We thank E. Nagao, K. Kinjoh. M Namisato, M. Goto, A. Hosokawa, T. Yanagihashi, R. Nogami, A.T. Agdamag and I. Agsuni.
PM & BM 13
EVALUATION OF GENETIC VARIABILITY IN Mycobacterium leprae AND POSSIBLE APLICATION FOR DEVELOPMENT OF MOLECULAR TOOLS FOR STRAIN TYPING
Fontes A.N.B .1, Brandão A.A.2, Santos A.R.2, Baptista I.M.F.D.1, Miranda A.B.1, Cho S.-N.3, Sarno E.N.2 and Suffys P.N.1
1Department of Biochemistry and Molecular Biology, Oswaldo Cruz Institute, Fiocruz, Avenida Brasil 4365, Manguinhos 21045-900, Rio de Janeiro, Brazil:
2Department of Tropical Medicine, Oswaldo Cruz Institute;
3Department of Microbiology, Yonsei University College of Medicine, 134 Shinchon-dong, Deodaemoon-ku, Seoul 120-752, Republic of Korea
Despite a considerable reduction in registered leprosy cases over the last 15 years, the disease is still a major public health problem in several countries with no substantial decrease in case detection rate. Control programs could be improved by identification of the source of infection, better understanding of transmission and if relapse cases could be differentiated from re-infection.
Attempts to identify individual strains of Mycobacterium leprae has so-far been disappointing and development of fingerprinting technology is hampered by the lack of DNA polymorphism. Very recently however, one study demonstrated a considerable isolateassocialed difference in the number of copies in a TTC repeat in a single locus.
We confirmed the difference in TTC copy number in this locus in M. leprae from different Brazilian leprosy patients using gel electrophoresis and automatic sequencing. After analyzing the M. leprae genome sequence for simple repeats, sets of primers for amplification of five more loci containing (AT)n , (TAC)n or (C)n-(G)n were developed. Our preliminary data, using skin biopsy samples from 3 different patients and agarose gel electrophoresis, demonstrated size variability in 3 of the 5 PCR systems so at the moment, 4 loci have been defined containing isolate-associated polymorphism. Considering the limited resolution of agarose, variability in the other 2 systems will be searched for on polyacrylamide gel and by sequencing. More samples are being collected, including biopsy and lymph samples from multi- and paucibacillary patients, in order to establish the degree of genetic variability in the different loci and look for a possible association between genetic composition of the bacilli using these markers and clinical and epidemiological characteristics of the patients.
PM & BM 14
FURTHER STUDIES ON THE HISTOLOGICAL CHANGES IN THE SKIN IN PRIMARY NEURITIC LEPROSY
Sujai Suneetha, Rajgopal Reddy, Suman Jain and Lavanya Suneetha
LEPRA India - Blue Peter Research Centre, Cherlapally, Hyderabad-501301
Primary neuritic leprosy presents with peripheral nerve damage and no evident skin patches. Clinical diagnosis has centred on demonstration of anaesthesia and nerve enlargement. Laboratory confirmation is based on a histological diagnosis of leprosy in the cutaneous nerve biopsy. We have previously shown that although the skin shows no visible patches there are histological evidences of the disease in the skin biopsy.
In the present study 24 PNL cases were subjected to a skin biopsy from the area of sensory change to look for any histological evidences of leprosy. 18 of the 24 biopsies (75%) showed changes specific to leprosy. The changes ranged from Indeterminate leprosy in 7 (29.2%), Indeterminate -> BT leprosy in 2 (8.3%). Indeterminate to BL leprosy in 3 (12.5%), BT in 5 (20.8%) and BL in 1 (4.2%). 6 of the biopsies revealed no significant lesion. The histological features suggest a schematic progression of the disease from non-specific changes to specific changes such as indeterminate leprosy and further progression to determined forms of BT and BL leprosy.
This gives PNL an important status as a stage in the development of full blown disease.
PM & BM 15
IMPROVED PROCEDURES FOR THE GENERATION OF THE RECOMBINANT ANTIGENS OF M. leprae
Hee Jin Kim, John S. Spencer, Maria Angela M. Marques, Monica C. B. S. Lima and Patrick J. Brennan
Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1677. U.S.A.
To date, most developmental work for new leprosy diagnostic antigens has been conducted on native M. leprae products. Since the M. leprae genome sequence has become available, emphasis has shifted to individual proteins, notably those that are M. leprae specific, For the purpose of producing recombinant antigens, we have modified standard methods. The basic 3-step strategy is well established: PGR. cloning of the genes into an expression vector, and purification of six-histidine-tagged proteins using the standard immobilized metal affinity column (IMAC). However, in order to produce large quantities of soluble recombinant proteins, we have modified these methods. We use touchdown PCR to overcome high annealing temperatures for high GC-rich DNA. In purifying recombinant proteins, we modify the pH gradient buffer system using the knowledge of pi values of the 6-histidine tag and IMAC. In this way, several soluble recombinant proteins have been produced in milligram quantities per one liter of cultured cells. The hydrophobicity and pi values of the original proteins determine the solubility and quantity purified. Ten M. leprae recombinant antigens (ESAT-6; CFP-10; MMP-I; MMP-II; EF-Tu: Ag85B: and the Ag85B+ESAT-6, CFP-10+ESAT-6. l0kDa+ ESAT-6 fusion proteins) were purified by these methods and are under investigation (supported by a grant and contract from the NIAID, NIH).
PM & BM 16
INTRACELLULAR SIGNALS TRIGGERED DURING ASSOCIATION OF Mycobacterium leprae WITH SCHWANN CELLS
Alves, L., Amaral, V.W.A and Pessolani, M.C.V.
Laboratório de Hanseníase, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Br.
Interaction of bacterial pathogens with their host cells triggers signal transduction pathways that, in turn, leads to a variety of cellular responses which ultimately favor their perpetuation in the host. Among these responses are those given rise to an extensive reorganization of the cytoskeleton. which results in morphological changes, and the secretion of cytokines into the medium. Although this interference in host cell metabolism by bacteria represents a central feature of their pathogenesis, these events are poorly understood in Schwann cells (SCs) infected with Mycobacterium leprae, the causative agent of leprosy. To gain a better understanding of M. leprae-SC interaction, the present study investigates the signal transduction events triggered during the interaction of M. leprae with SCs. The assays consiste of pre-treating or not ST88-14 cells -a human Schwann cell line - with specific kinase inhibitors, followed by incubation with fluorescein-labeled bacteria and analysis of bacterial association via fluorescence microscopy. The use of tyrphostin AG 126, bisindolylmaleimide I and wortmannin which, respectively, inhibit tyrosine kinase (TK), protein kinase C (PKC) and phosphalidylinositol 3- kinase (PI 3-K) produced association inhibition suggesting that TK. PKC and PI 3-K are activated during the interaction of the leprosy bacillus with SC. Currently these preliminary results are being confirmed and the involvement of other transduction elements are being investigated by the use of specific inhibitors.
Supported by PAPES 2/ FIOCRUZ, the Heiser Program for Reseach in Leprosy and Tuberculosis and CAPES.
PM & BM 17
INTRACELLULAR TRAFFICKING OF Mycobacterium leprae IN SCHWANN CELLS
Leila de Mendonça Lima1, Maria Cristina Vidal Pessolani2, Euzenir Nunes Sarno2 and Lucia P. Barker3
1Dept. of Biochemistry and Molecular Biology and 2Leprosy Laboratory, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil; 3Dept. of Medical Microbiology and Immunology, School of Medicine, Univ. of Minnesota, Duluth, MN, USA.
The effects of M. leprae invasion on the physiology and metabolism of Schwann cells and its relation to the progressive and irreversible degenerative process of peripheral nerves are poorly understood. M. leprae almost exclusively infects macrophages and Schwann cells. The fate of other pathogenic mycobacteria, once inside macrophages, has been the object of many recent studies. The exact molecular events leading to the M. tuberculosis phagosome-lysosome fusion inhibition, initially identified by Armstrong and Hart (J Exp Med 134:713-740, 1971). are not yet completely understood, though great progress has been made in the delineation of the molecules involved in uptake of M. tuberculosis and its interference with fundamental trafficking processes in host cells. Earlier studies, using bone-marrow derived macrophages, have shown that, like other pathogenic mycobacteria. M. leprae resides in non-acidified phagosomes (Frehel and Rastogi. Infect Immun 55: 2916-2921, 1987).
We have used a human Schwannoma cell line (ST 8814) as an in vitro model for M. leprae infection. Tissue culture cells were incubated with fluorescently labeled live and heat-killed M. leprae (kindly provided by J. Krahenbuhl, Louisiana State Univ., Baton Rouge. Louisiana, USA). Our data demonstrate that the cells avidly take up both live and dead M. leprae. Further, in preliminary experiments using fluorescent markers of lysosomal compartments, we show that live M. leprae do not collocalize with acidified vesicles inside Schwann cells, whereas dead bacilli do. This is the first demonstration of the utility of the ST 8814 cell line to study the trafficking of M. leprae in vitro. Studies are under way to further characterize the intracellular fate of M. leprae in Schwann cells.
This work received financial support from WHO/TDR and the Brazilian Ministry of Health
PM & BM 18
LEPROSY TRANSMISSION AND MUCOSAL IMMUNE RESPONSE: DO SEASONS PLAY ANY ROLE?
Dr. R.S. Jadhav, Miss A. Fernando, Miss V.S. Shinde, Ravindra R. Kamble, Mrs. S.P. Madhale, Dr. V.K. Edward, Dr. J.R. Rao and Prof. W.C.S. Smith on behalf of MILEP-2 Study Group*
Stanley Browne Research Laboratories, Richardson Leprosy Hospital, Miraj, Maharashtra-416410, Tel. No Off: 0233-211213 Fax: 0233-211708 E-mail: shlabtlm@vsnl.com
Environmental association with the incidence of leprosy is not yet well understood. Reports from the literature indicate association of the rainfall and proximity of water sources to the inhabitants with the incidence. This can be an important aspect in the transmission of leprosy, especially in reference to the reports that suggest the increased viability of M. leprae outside human body under moist and shaded conditions. The objective of the present analysis was to look at the presence of M. leprae on the nasal mucosa in general population in different seasons using the data obtained from the study that was designed to look at the transmission and the development of mucosal immunity. Individuals from three villages were screened. Polymerase Chain Reaction (PCR) was used to detect presence of M. leprae DNA on the nasal mucosa and mucosal immunity was tested by measuring the salivary M. leprae reactive IgA antibodies (sML-IgA) using ELISA. PCR positivity was seen to be highest during the monsoon season. The PCR positivity was seen in 2.5% (36 out of 1464), 1% (19 out of 1824) and 4% (68 out of 1701) subjects during winter, summer and monsoon seasons respectively. Both children and adults show peak of positivity in the monsoon suggesting an increased exposure to M. leprae in monsoon. The percentage of non-exposed subjects i.e. subjects negative for PCR and sML-IgA is highest in summer (37.9%) and lowest in monsoon (27.4%). Seasonal effect and dynamic nature of the exposure needs to be looked more closely with shorter duration follow-ups to understand the mechanism of transmission and factors affecting it, which in turn can help us to design intervention strategies to interrupt the transmission.
PM & BM 19
MAST CELL SUBSETS AND NEUROPEPTIDES IN LEPROSY REACTIONS
Sérgio Luiz Gomes Antunes, Yong Liang, José Augusto da Costa Neri, Euzenir Nunes Sarno, Mary Haak-Frendscho, Olle Johansson
Oswaldo Cruz Institute, Laboratory of Leprosy, Rio de Janeiro, Brazil, The Experimental Dermatology Unit, Department of Neuroscience, Karolinska Institute, Stockholm, Sweden, Promega Corporation, Madison, WI, USA
The immunohistochemical identification of neuropeptides (calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P, -melanocyte stimulating hormone and -melanocyte stimulating hormone) quantification of mast cells and their subsets (tryptase/chymase-immunoreactive mast cells = TCMC and tryptase-immunoreactive mast cells = TMC) were determined in biopsies of six patients with leprosy reactions (three patients with type I reaction and three with type II). Biopsies were compared with those taken from the same body site in the remission stage of the same patient. We found a relative increase of TMC in the inflammatory infiltrate of the reactional biopsies compared to the post-reactional biopsy. Also, the total number of mast cells and the TMC/TCMC ratio in the inflammatory infiltrate was significantly higher than in the intervening dermis of the biopsies of both periods. No significant difference was found regarding neuroptide expression in the reactional and post-reactional biopsies. The relative increase of TMC in the reactional infiltrates could implicate this mast cell subset in the reported increase of the immune response in leprosy reactions
PM & BM 20
MORPHOLOGICAL EVALUATION OF NERVE BIOPSIES FROM PURE NEURITIC FORMS OF LEPROSY USING TOLUIDINE BLUE-STAINED SEMITHIN SECTIONS. CORRELATION WITH THE RESULTS OF POLYMERASE CHAIN REACTION
Antunes. S.L.G.; Rodrigues, M.J.; Santos, A.R.; Chimelli. L.M.; Faria, C.R.S.; Fernandes, P.V.; Sarno, E.N.
Fundação Oswaldo Cruz, Universidade Federal do Rio de Janeiro
Pure neuritic leprosy is difficult to diagnose if acid-fast baccilli is neither detected in nerve biopsy sections nor in skin smears. Nerve biopsies of seventeen patients with neuritic form of leprosy were submitted to the routine histopathology (H-E and Wade staining) and also to semi-thin (0,5 m) sectioning, toluidine blue staining and were observed under optical microscopy. A small piece of the nerve biopsy was submitted to polymerase chain reaction (PCR) for the detection of M. leprae DNA. The morphological findings of the biopsies were: inflammatory infiltrate (9), fibrosis (8, six of them with concomitant inflammatory process), myelinated liber loss (13, large or small fibers), demyelination (3), active axonal degeneration: (2), remyelinalion (7), axonal regeneration (4). endoneurial angiogenesis and nuiltilayering of capillary wall (5), acid-fast baccilli posilivity (5). Eleven biopsies were PCR-positive. (6 of them were AFB-negative in Wade staining and one of them exhibited normal histological appearance). The most predominant findings for leprosy neuriiic form were perineurijm and endoneurium inflammatory infiltrate, nerve fiber loss (small and large libers) and fibrosis. PCR contributed decisively in 6 cases for the diagnosis
PM & BM 21
MULTIPLE ENDOTHELIAL MEMBRANE PROTEINS BIND M. leprae
D.M. Scollard, G.T. McCormick, and TP. Gillis
Laboratory Research Branch, National Hansen's Disease Program at LSU, Baton Rouge, LA.
Morphologic evidence has suggested that endothelial cells (EC) may be the gateway through which M. leprae enter peripheral nerve. Studies in vitro have demonstrated that uptake of M. leprae by EC is time-and dose-related. Experiments have therefore been undertaken to identify the EC membrane proteins capable of binding M. leprae.
Cytoplasmic membranes from 12 x 106 EC grown in vitro were solubilized and their proteins conjugated to biotin. M. leprae (2 x 109) were allowed to bind these bioiinylaled proteins for 4 hr at 4º C. The bacterial pellet was washed to remove unbound proteins: bound proteins were separated by SDS-PAGE and electro-transferred to PVDF membranes. Biotinylated EC proteins were visualized by staining with an avidin-alkaline phosphatase conjugate.
Biotinylated EC proteins bound to M. leprae were separated into several distinct bands, 7 of which have been consistently identified in 8 different experiments. In these preliminary experiments, the smaller molecules (29, 32, 47, and 54 kDa) have yielded discrete single bands on 8% and 10% gels; the larger molecules have appeared more diffuse, with bands at 59-63, 125-130. and 175-185 kDa.These studies suggest that EC are capable of binding M. leprae using multiple surface proteins. Although these probably include proteins already used by other cell types to M. leprae, they may also include binding proteins unique to EC.
PM & BM 22
NEURAL PREDILECTION. MOLECULAR MIMICRY AND NERVE DAMAGE- COMPUTATIONAL COMPARISONS BETWEEN M . leprae BINDING PROTEINS AND THE M . leprae GENOME
Deena Vardhini, Lavanya M, Suneetha, Niyaz Ahmed* and Sujai K Suneetha
Lepra India - Blue Peter Research Centre, Cherlapally, Hyderabad 501 301.
*Centre for DNA Fingerprinting and Diagnostics, Nacharam, Hyderabad.
Mycobacterium leprae, the causative organism of leprosy is known to target and infect Schwann cells of the human peripheral nerve and trigger host-mediated immune reactions and destruction of myelin membranes leading to nerve damage. Antigenic mimicry is a mechanism adopted by M. leprae to evade the efficient human immune system. Various receptor mediated mechanisms such as the laminin 2-α dystroglycan/β integrin bridge, the libronectin (FN)-β integrin bridge and the myelin P0 glycoprotein are known to play a role in the binding and invasion of Schwann cells by M. leprae. Computational comparison of the M. leprae proteins and the human peripheral nerve - M. leprae binding proteins has revealed sequence similarities. Laminin had a homology to 60 kDa Chaperon in 1 and heat shock protein (P values 0.55 and 0.94 respectively).
Fasta searches of libronectin and Blastp searches of myelin P0 revealed a homology to the secreted P60 family protein. The significance of secreted proteins as antigenic determinants is of importance because in tuberculoid leprosy nerve pathogenesis is observed even in the absence of M. leprae.
The secreted P60 family protein has sequence similarities to the immunoglobin domains of myelin P0. which have significance in protein-protein and protein-ligand interactions. FSSP studies showed that many of the structural neighbours of myelin P0 were antigenic determinants and/or immunogens, which could have implications in understanding nerve damage.
These sequence similarities need to be further analyzed by extending this bioinformatic knowledge to wet experimentation to recognize potential drug tar-gels and peptides to counteract leprosy.
PM & BM 23
OBSERVATION OF ACID FAST BACILLI BY MERGE TECHNIQUE OF DIFFERENTIAL INTERFERENCE CONTRAST AND POLARIZED MICROSCOPES
Furuta Mutsuhiro1. Hatano Kentaro2, Matsuki Takanobu2, Okano Yoshiko2, Ikeda Takeshi3, Nakatani Koichi3 and Sato Atsuo3
1Izumigaoka Hospital,
2National Sanatorium Oku-Komyo-En 3National Minami Kyoto Hospital
Approximately 400 autopsy cases of leprosy were done during past 50 years by our group. There are still many unsolved problems in the pathological field of the acid fast bacillary infections.
We have studied the polarization of M. leprae under the polarized microscope up to a thousand magnification in these six years and trying to extend this study to the other mycobacterial infections such as tuberculosis and MOTT (Mycobacterium Other Than Tuberculosis). The polymorphonuclear leukocytes respond to these mycobacterium in the first phase of infection, then later phagocytic histiocytes take place of the role of responder. Polarization of mycobacterium was not observed in the polymorphonuclear leukocytes and monocyts in early stage when acid fast bacillary stains clearly. On the contrary, after the acid fast bacillary stain become negative in the late stage of treatment, polarized particles similarly looking to mycobacterium come appear in the cytoplasma of phagocytic histiocytes. Later on, the polarized mycobacteria are seen in the surrounding collagenous connective tissue. We will try to investigate these polarized particles using marge technique of differential interference contrast.
These sequence similarities need to be further analyzed by extending this bioinformatic knowledge to wet experimentation to recognize potential drug targets and peptides to counteract leprosy.