Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Molecular cloning of alpha antigen like protein gene of Mycobacterium leprae and its over production in Escherichia coli

I have constructed the genomic library of M. leprae Thai 53 strain, and cloned the alpha antigen like protein gene by plaque hybridization method by using M. leprae alpha antigen DNA fragment as probe which was characterized in the previous study, I have termed it as alpha 2 antigen gene. The alpha 2 antigen gene has been characterized by sequencing. By comparing the deduced amino acid sequence of alpha and alpha 2 antigen with 85 complex antigen of other mycobacteria. I have found the higher homology between alpha 2 antigen and 85A antigen and between alpha antigen and 85B antigen. We have constructed the over expression system of M. leprae alpha and alpha 2 antigen gene in E. coli using vector pMALc-RI. Recombinant alpha and alpha 2 antigen has been purified by amylose column chromatography at the purity of more than 95%. More than 6 mg and more than 10 mg of recombinant alpha and alpha 2 antigen has been obtained from 200 ml of liquid culture, respectively. ELISA tests have been performed with the sera of leprosy patient and healthy control against the recombinant alpha and alpha 2 antigens. The antibody titers in sera of leprosy patient against the two kinds of antigens were all much higher than healthy controls. The antibody titer against the alpha 2 antigen was higher than that against alpha antigen. Recombinant alpha and alpha 2 antigens in this study could be used as a new specific antigen for serodiagnosis of leprosy.     

Activity of two doses of rifampin against Mycobacterium leprae

In the course of a clinical trial designed to re-examine the bactericidal efficiency of 600-mg doses of rifampin (RMP) against Mycobacterium leprae, two doses of RMP, either 600 mg or 1200 mg, were administered 28 days apart to 29 previously untreated patients with lepromatous or borderline leprosy. Seven, 28, and 35 days after the start of the trial, skin biopsies were performed and immunologically normal mice were inoculated with 5 x 10(3) or 10(4) M. leprae in each hind foot pad. The patients assigned to the two regimens did not differ significantly in terms of sex, age, disease classification, bacterial index, or the concentration of M. leprae in the skin lesion biopsied for the inoculation of mice. The concentrations of organisms in the skin-biopsy specimens did not change significantly over the course of the trial among the patients, whether they were being treated by the first or the second regimen. The M. leprae recovered from specimens obtained from 21 of the patients, before beginning treatment, multiplied in a majority of the mice inoculated. The results of mouse inoculation confirmed the rapid bactericidal effects of RMP against M. leprae: a single dose of RMP rendered the organisms obtained from all but two of the patients incapable of multiplying in mice. No significant difference was demonstrated between the two regimens, nor was an additional effect of the second dose of RMP observed.     

Homologs of Mycobacterium leprae 18-kilodalton and Mycobacterium tuberculosis 19-kilodalton antigens in other mycobacteria

Most of the antigens of Mycobacterium leprae and M. tuberculosis that have been identified are members of stress protein families, which are highly conserved throughout many diverse species. Of the M. leprae and M. tuberculosis antigens identified by monoclonal antibodies, all except the 18-kDa M. leprae antigen and the 19-kDa M. tuberculosis antigen are strongly cross-reaction between these two species and are coded within very similar genes. Studies of T cell reactivity against mycobacterial antigens have indicated that M. tuberculosis bears epitopes that are cross-reactive with the M. leprae 18-kDa antigen, but attempts to identify an 18-kDa antigen-like protein or protein coding sequence in M. tuberculosis have been unsuccessful. We have used a combination of low-stringency DNA hybridization and polymerase chain reaction techniques to identify, isolate, and sequence genes from M. avium and M. intracellulare that are very similar to the 18-kDa antigen gene of M. leprae and others that are homologs of the 19-kDa antigen gene of M. tuberculosis. Unlike M. leprae, which contains a single 18-kDa antigen gene, M. avium and M. intracellulare each have two 18-kDa antigen coding sequences. Although the M. leprae, M. avium, and M. intracellulare 18-kDa antigen genes are all very similar to one another, as are the M. tuberculosis, M. avium, and M. intracellulare 19-kDa antigen genes, we have been unable to detect any 18-kDa antigen-like coding sequences in DNA from M. tuberculosis.     

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.     

Detection of IgA anti-PGL-I specific antigen to Mycobacterium leprae in mangabey monkeys inoculated with M. leprae

OBJECTIVE: This study examined the reliability of the three-cluster model for chronic low back pain patients found using the Integrated Psychosocial Assessment Model (IPAM). A replication study using a sample of patients from a different country was completed. PATIENTS: Seventy patients (average age = 47.05 years, SD = 16.11) with chronic low back pain of noncancer origin participated in the study. Sixty-two of these patients were attending The Auckland New Zealand Regional Pain Service, while a further eight were attending a private practice pain service in Auckland. OUTCOME MEASURES: Subjects were assessed on the IPAM, which measures pain intensity, disability, coping strategies, attitudes towards and beliefs about pain, depression and illness behaviour, the Medical Examination and Diagnostic Information Coding System, and the Multidimensional Pain Inventory. RESULTS: Cluster analyses using the kappa-means algorithm were performed on the IPAM data. The three-cluster solution was preferred according to both the Variance Ratio Criterion and cluster interpretability. Two of the three clusters correlated highly with clusters retrieved in the original study (r = 0.78, r = 0.71), while the third cluster showed partial resemblance (correlation of r = 0.31). Clusters were named "In Control," "Depressed and Disabled," and "High Deniers and Somatizisers." No differences were found on the physical pathology scores between clusters. Decision rules for cluster assignation resulted in 68% of the sample being correctly assigned. CONCLUSIONS: Support for this cluster model from two countries suggests its value in providing a multidimensional picture of patients with chronic low back pain. The possibility of using such cluster groups for determining treatment type is discussed.     

Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M. leprae protein antigens, and, with emphasis on recent developments, summarize what is known regarding their functional and immunological features.     

Role of alpha-dystroglycan as a Schwann cell receptor for Mycobacterium leprae

alpha-Dystroglycan (alpha-DG) is a component of the dystroglycan complex, which is involved in early development and morphogenesis and in the pathogenesis of muscular dystrophies. Here, alpha-DG was shown to serve as a Schwann cell receptor for Mycobacterium leprae, the causative organism of leprosy. Mycobacterium leprae specifically bound to alpha-DG only in the presence of the G domain of the alpha2 chain of laminin-2. Native alpha-DG competitively inhibited the laminin-2-mediated M. leprae binding to primary Schwann cells. Thus, M. leprae may use linkage between the extracellular matrix and cytoskeleton through laminin-2 and alpha-DG for its interaction with Schwann cells.     

Immunohistochemical study on temporal bone of autopsy cases with Mycobacterium leprae infection

Three temporal bones were obtained en bloc from autopsy cases with lepromatous leprosy from the middle cranial fossa side after removing the brain. After fixation with 10% formalin followed by sufficient decalcification, the specimens were embedded in paraffin en bloc and cut serially to stain every 10th section with hematoxylin and eosin (H&E) for anatomical orientation. An immunohistochemical study with anti-neurofilament, anti-MBP (myelin basic protein), anti-BCG (Bacillus Calmette-Gue'rin) and anti-PGL (Mycobacterium leprae-specific antiphenolic glycolipid-I) antibodies were performed to vestibular, cochlear and facial nerves, respectively, on the basis of anatomical orientation of the adjacent H&E sections. In one of three cases, positive staining by anti-PGL antibodies was recognized only in the facial nerve both in its internal auditory meatal and tympanic portions. However, even in this case, no neural damage was observed either by anti-neurofilament nor anti-MBP stainings. This finding supports the possibility of central neural infection by Mycobacterium leprae.     

Factors influencing the in vitro growth of Mycobacterium leprae: effect of inoculum

Factors responsible for the in vitro growth of Mycobacterium leprae in Dhople-Hanks (DH) medium, and also to improve the technique devised earlier, and the source of the M. leprae used as inoculum, were investigated. M. leprae were obtained from armadillos and nude mice, both inoculated earlier with human- or armadillo-derived M. leprae. The growth of M. leprae in DH medium was monitored using two biochemical indicators. Normal growth was obtained when inocula were from livers and spleens of M. leprae-infected armadillos. The M. leprae harvested from the footpads of nude mice failed to multiply in the same medium. Using inocula from livers and spleens of infected armadillos, a gradual decrease in inoculum size resulted in a proportionately slower multiplication of M. leprae. When the DH medium was supplemented with whole M. leprae, or cell-free extracts of M. leprae, from irradiated livers and spleens of infected armadillos, nude mouse-derived M. leprae exhibited growth in the DH medium in accord with that obtained using armadillo-derived M. leprae. Similar results were obtained with cell-free extracts of M. leprae harvested from non-irradiated livers and spleens of infected armadillos, but no growth was obtained when the medium was supplemented with extracts from livers or spleens of normal armadillos. These results indicate the possible existence of a growth factor in armadillo-derived M. leprae.     

Mycobacterium leprae binds to a 25-kDa phosphorylated glycoprotein of human peripheral nerve

Mycobacterium leprae, the causative agent of leprosy, specifically invades and destroys the peripheral nerve, which results in the main clinical manifestation of the disease. Little is known about the bacteria-nerve protein interaction. We show in the present work that M leprae binds to a 25 kDa glycoprotein from human peripheral nerve. This protein is phosphorylatable and it binds to lectins which have alpha-mannose specificity. This M leprae-protein interaction could be of importance in the pathogenesis of leprosy.     

Lipids from Mycobacterium leprae cell wall suppress T-cell activation in vivo and in vitro

The need for an ethics of medical justice in Latin America is asserted in the context of a review of concepts of justice throughout history and of changing governmental perspectives on provision of health care in the US and other developed countries. The current view that individuals are primarily human resources is at odds with a long tradition asserting the intrinsic dignity of human beings. English-speaking bioethicists began in the 1960s to stress the principal of autonomy of patients, recognizing their right to make decisions on their own lives and medical care equally with the physician. At the same time, the US has approved no legislation establishing a right to health care, which is rather regarded as a private good. Governments are increasingly inclined to renounce their role as direct providers of health care. The liberal democratic state until recently understood that it fulfilled its ethical commitment to promoting social justice through provision of health care. Nevertheless, societies that stress the importance of the individual in decision-making and that conceive of health as a private good are confronted with the contradiction of apparently irreconcilable visions. With infinite demand for health services and limited health resources, the discourse of autonomy has slowly been replaced by a discourse of distributive justice. The most appropriate version of distributive justice for Latin America is probably that which affirms the duty of assisting those most in need. The prevalence of malnutrition, misery, and premature death in the world is a clear sign of imbalance. If the essential dignity of all human beings and not just of the elite is to be affirmed, medical justice must become the most urgent priority of Latin America.     

Use of an ordered cosmid library to deduce the genomic organization of Mycobacterium leprae

In an attempt to unify the genetic and biological research on Mycobacterium leprae, the aetiological agent of leprosy, a cosmid library was constructed and then ordered by a combination of fingerprinting and hybridization techniques. The genome of M. leprae is represented by four contigs of overlapping clones which, together, account for nearly 2.8Mb of DNA. Several arguments suggest that the gaps between the contigs are small in size and that virtually complete coverage of the chromosome has been obtained. All of the cloned M. leprae genes have been positioned on the contig maps together with the 29 copies of the dispersed repetitive element, RLEP. These have been classified into four groups on the basis of differences in their organization. Several key housekeeping genes were identified and mapped by hybridization with heterologous probes, and the current genome map of this uncultivable pathogen comprises 72 loci.     

Characterization of circulating immune complexes in leprosy patients and their correlation with specific antibodies against Mycobacterium leprae

Thirty patients between the ages of 7 months and 24 years were treated surgically for symptomatic Chiari II malformation at the Arkansas Children's Hospital. All patients underwent at least bony decompression of the malformation. Assessments of the patients' conditions were made at 6 weeks and 1 year after surgery, and complications of surgery were noted. For a majority of the patients, the presenting symptoms were resolved following treatment (74% at 6 weeks and 80% at 1 year). Partial resolution occurred in several of the patients (17% at 6 weeks and 14% at 1 year). A small number remained the same at 6 weeks (6%) and at 1 year (3%), while 1 patient was worse after surgery. Ten of the patients with complete resolution in the short term required repeat surgery an average of 49 months after their original operation, after which they again attained complete resolution of their symptoms. Scales for clinical, radiographic and operative grading of the patients' conditions as mild, moderate or severe were devised, and these were employed to characterize the condition of each patient. Patients in each grading category had good results, with rates of complete symptomatic resolution ranging from 67 to 100%. Severity in each category was found to be well correlated with eventual recurrence of symptoms and need for reoperation.     

Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen

In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp. The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco. bovis (bacille Calmette-Guérin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts. In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay. Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco. leprae hsp70-reactive individuals responded to C-344. Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344. The smaller C-142 fragment which includes the terminal 70 residues unique to Myco. leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees. In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared. Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes. The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides. Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes. Although both were located in regions of the molecule shared with Myco. tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.