Humulus japonicus rescues autistic­like behaviours in the BTBR T+ Itpr3tf/J mouse model of autism.

Humulus japonicus (HJ) is a traditional herbal medicine that exhibits anti­inflammatory, antimicrobial and anti­tumor effects that is used for the treatment of hypertension, pulmonary disease and leprosy. Recently, it has also been reported that HJ demonstrates neuroprotective properties in animal models of neurodegenerative diseases. The current study hypothesised that the administration of HJ would exhibit therapeutic effects in autism spectrum disorder (ASD), a neurodevelopmental disorder with lifelong consequences. The BTBR T+ Itpr3tf/J mouse model of ASD was used to investigate the anti­autistic like behavioural effects of HJ. Chronic oral administration of the ethanolic extract of HJ significantly increased social interaction, attenuated repetitive grooming behaviour and improved novel­object recognition in BTBR mice. Anti­inflammatory effects of HJ in the brain were analysed using immunohistochemistry and reverse­transcription quantitative PCR analysis. Microglia activation was markedly decreased in the striatum and hippocampus, and pro­inflammatory cytokines, including C­C Motif Chemokine Ligand 2, interleukin (IL)­1ß and IL­6, were significantly reduced in the hippocampus following HJ treatment. Moreover, HJ treatment normalised the phosphorylation levels of: N­methyl­D­aspartate receptor subtype 2B and calcium/calmodulin­dependent protein kinase type II subunit α in the hippocampus of BTBR mice. The results of the present study demonstrated that the administration of HJ may have beneficial potential for ameliorating behavioural deficits and neuroinflammation in ASD.

Komagataeibacter hansenii CGMCC 3917 alleviates alcohol-induced liver injury by regulating fatty acid metabolism and intestinal microbiota diversity in mice.

The potential effects of Komagataeibacter hansenii CGMCC 3917 cells on alcohol-induced liver injury and their probable mechanisms were investigated. Male Kunming mice were orally administered with alcohol (10 mL per kg BW) alone or in combination with administration of K. hansenii CGMCC 3917 cells at 2 × 108 and 2 × 106 CFUs for 10 weeks. Administration of strain CGMCC 3917 cells, especially high dose administration, decreased the liver weights, fat gain, and fatty-acid metabolism-related enzyme SCD-1, ACC and FAS expressions and endotoxin release, which were elevated by alcohol treatment. Furthermore, the total contents of long chain fatty acids of the liver and serum in alcohol-treated mice supplemented with a high dose of strain CGMCC 3917 cells were decreased to 5.44 ± 0.19 µg mL-1 and 3.66 ± 0.15 µg mL-1 from 6.65 ± 0.31 µg mL-1 and 4.52 ± 0.21 µg mL-1, respectively. Conversely, the SCFAs decreased by ethanol treatment, particularly the acetic acid, propionic acid and butyric acid, were obviously enhanced in the faeces, colon and cecum of the mice supplemented with strain CGMCC 3917 cells. Moreover, strain CGMCC 3917 cells could regulate gut microbiome by significantly decreasing the abundance of Actinobacteria, Proteobacteria and Firmicutes, and dramatically increasing the abundance of Bacteroidetes in alcohol-treated mice. These findings suggest that K. hansenii CGMCC 3917 cells alleviate alcohol-induced liver damage via regulating fatty acid metabolism and intestinal microbiota diversity in mice.

A Mycobacterium leprae Hsp65 mutant as a candidate for mitigating lupus aggravation in mice.

Hsp60 is an abundant and highly conserved family of intracellular molecules. Increased levels of this family of proteins have been observed in the extracellular compartment in chronic inflammation. Administration of M. leprae Hsp65 [WT] in [NZBxNZW]F(1) mice accelerates the Systemic Lupus Erythematosus [SLE] progression whereas the point mutated K(409)A Hsp65 protein delays the disease. Here, the biological effects of M. leprae Hsp65 Leader pep and K(409)A pep synthetic peptides, which cover residues 352-371, are presented. Peptides had immunomodulatory effects similar to that observed with their respective proteins on survival and the combined administration of K(409)A+Leader pep or K(409)A pep+WT showed that the mutant forms were able to inhibit the deleterious effect of WT on mortality, indicating the neutralizing potential of the mutant molecules in SLE progression. Molecular modeling showed that replacing Lysine by Alanine affects the electrostatic potential of the 352-371 region. The number of interactions observed for WT is much higher than for Hsp65 K(409)A and mouse Hsp60. The immunomodulatory effects of the point-mutated protein and peptide occurred regardless of the catalytic activity. These findings may be related to the lack of effect on survival when F(1) mice were inoculated with Hsp60 or K(409)A pep. Our findings indicate the use of point-mutated Hsp65 molecules, such as the K(409)A protein and its corresponding peptide, that may minimize or delay the onset of SLE, representing a new approach to the treatment of autoimmune diseases.

Ovarian follicle loss in humans and mice: lessons from statistical model comparison.

BACKGROUND: Mammalian oocyte stocks reach maximum size in early development and begin depletion immediately thereafter. This depletion ends women's fertility by midlife. Here we compare five models proposed to characterize human follicular depletion, highlight underlying variation in atresia, and use oocyte counts from laboratory mice to illustrate possible effects of known covariates. METHODS: We compared statistical models, of human data, from five well-known sources and also compared the models' fit to data from four genetically distinct strains of mice. RESULTS: A model first published by Hansen et al. (2008) fit the human data better than any of the alternatives. Best-fit models of oocyte loss in the four strains of mice differed substantially from the best-fit model of the aggregated mouse data. CONCLUSIONS: Although the power model published by Hansen et al. (2008) fit the human data best, Faddy and Gosden's (1996) differential equation model may be a more useful characterization of human follicular atresia. However, these models leave a great deal of variation unexplained. Mouse strain comparisons show that follicle loss in genetically distinct subpopulations can differ substantially from the pattern in the aggregate population. This indicates that differences in follicular stock size between and within populations depend upon more than a single predictor (i.e. age or follicle stocks at previous time points). Our reliance upon data from Western populations represents this study's most important limitation. Expanding data collection to include likely covariates and a wider range of human populations would improve the basis for predicting individual trajectories of follicle loss as more women worldwide opt to delay childbearing and risk aging beyond their own windows of fertility.

Expression of cyclooxygenase-2, alpha 1-acid-glycoprotein and inducible nitric oxide synthase in the developing lesions of murine leprosy.

Murine leprosy is a chronic disease of the mouse, the most popular animal model used in biomedical investigation, which is caused by Mycobacterium lepraemurium (MLM) whose characteristic lesion is the macrophage-made granuloma. From onset to the end of the disease, the granuloma undergoes changes that gradually transform the environment into a more appropriate milieu for the growth of M. lepraemurium. The mechanisms that participate in the formation and maturation of the murine leprosy granulomas are not completely understood; however, microbial and host-factors are believed to participate in their formation. In this study, we analysed the role of various pro-inflammatory and anti-inflammatory proteins in granulomas of murine leprosy after 21 weeks of infection. We assessed the expression of cyclooxygenase-2 (COX-2), alpha acid-glycoprotein (AGP), and inducible nitric oxide synthase (iNOS) at sequential stages of infection. We also looked for the nitric-oxide nitrosylation product, nitrotyrosine (NT) in the granulomatous lesions of murine leprosy. We found that a pro-inflammatory environment predominates in the early granulomas while an anti-inflammatory environment predominates in late granulomas. No obvious signs of bacillary destruction were observed during the entire period of infection, but nitrosylation products and cell alterations were observed in granulomas in the advanced stages of disease. The change from a pro-inflammatory to an anti-inflammatory environment, which is probably driven by the bacillus itself, results in a more conducive environment for both bacillus replication and the disease progression.

Development and application of rodent models for type 2 diabetes.

The increasing worldwide incidence of diabetes in adults constitutes a global public health burden. It is predicted that by 2025, India, China and the United States will have the largest number of people with diabetes. According to the 2003 estimates of the International Diabetes Federation, the diabetes mellitus prevalence in the USA is 8.0% and approximately 90-95% of diabetic Americans have type 2 diabetes - about 16 million people. Type 2 diabetes is a complex, heterogeneous, polygenic disease characterized mainly by insulin resistance and pancreatic beta-cell dysfunction. Appropriate experimental models are essential tools for understanding the molecular basis, pathogenesis of the vascular and neural lesions, actions of therapeutic agents and genetic or environmental influences that increase the risks of type 2 diabetes. Among the animal models available, those developed in rodents have been studied most thoroughly for reasons such as short generation time, inherited hyperglycaemia and/or obesity in certain strains and economic considerations. In this article, we review the current status of most commonly used rodent diabetic models developed spontaneously, through means of genetic engineering or artificial manipulation. In addition to these models, the Psammomys obesus, rhesus monkeys and many other species are studied intensively and reviewed by Shafrir, Bailey and Flatt and Hansen.

Immunoprophylaxis against Mycobacterium leprae infection with subunit vaccines.

We have investigated the effect of subunit vaccines against infection with Mycobacterium leprae, employing DNA plasmids as the vaccine vectors, and the immunodominant 35 kDa protein of M. leprae as the candidate antigen. A DNA vaccine that expresses the M. leprae 35 kDa protein both stimulated interferon-gamma (IFN gamma)-secreting T cells in mice, and demonstrated protection against M. leprae-infection of mice.

Combination of rifapentine-moxifloxacin-minocycline (PMM) for the treatment of leprosy.

To further the development of a multidrug regimen for treatment of leprosy that is suitable for monthly administration and fully supervisable, the bactericidal activities against Mycobacterium leprae of HMR 3647 (HMR), moxifloxacin (MXFX) and rifapentine (RPT) were measured by the proportional bactericide technique in the mouse footpad system, and compared with those of the established antileprosy drugs clarithromycin (CLARI), ofloxacin (OFLO) and rifampicin (RMP). Administered in five daily doses of 100 mg per kg body weight, HMR appeared slightly more bactericidal than CLARI, but the difference did not attain statistical significance. Administered as single doses, MXFX in a dosage of 150 mg per kg was more active than OFLO in the same dosage, and displayed the same level of activity as RMP in a dosage of 10 mg per kg; the combination MXFX-minocycline (MINO) (MM) was more bactericidal than the combination OFLO-MINO (OM); RPT in a dosage of 10 mg per kg was more bactericidal than RMP administered in the same dosage, and even more active than the combination RMP-OFLO-MINO (ROM); the combination RPT-MXFX-MINO (PMM) killed 99.9% of viable M. leprae, and was slightly more bactericidal than was RPT alone, indicating that the combination PMM showed an additive effect against M. leprae. These promising results justify a clinical trial among lepromatous patients, in which MM is being compared with OM, and PMM with ROM, in terms of efficacy and tolerance.

The Nramp1 protein and its role in resistance to infection and macrophage function.

Susceptibility to infectious diseases is under genetic control in humans. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. The Nramp1 gene was isolated by positional cloning the host resistance locus Bcg/Ity/Lsh, and mutations at this locus impair the resistance of mice to infections with intracellular parasites, such as Salmonella, Leishmania, and Mycobacterium. Allelic variants at the human Nramp1 homologue have recently been found to be associated with susceptibility to tuberculosis and leprosy in humans. The Nramp1 protein is an integral membrane protein expressed exclusively in the lysosomal compartment of monocytes and macrophages. After phagocytosis, Nramp1 is targeted to the membrane of the microbe-containing phagosome, where it may modify the intraphagosomal milieu to affect microbial replication. Although the biochemical mechanism of action of Nramp1 at that site remains unknown, Nramp homologues have been identified in many other animal species and actually define a protein family conserved from bacteria to humans. Some of these homologues have been shown to be divalent cation transporters. Recently, a second member of the mammalian Nramp family, Nramp2, was discovered and shown to be mutated in animal models of iron deficiency. The Nramp2 protein was subsequently shown to be the major transferrin-independent iron uptake system of the intestine. Together, these results suggest that Nramp1 may control intracellular microbial replication by actively removing iron or other divalent cations from the phagosomal space.

Predominant recognition of species-specific determinants of the GroES homologues from Mycobacterium leprae and M. tuberculosis.

The Mycobacterium leprae and M. tuberculosis 10,000 MW heat-shock protein homologues of GroES have previously been identified as major immunogens for human T cells. We used synthetic peptides to characterize the determinants recognized by murine T cells. The findings suggest that, despite 90% sequence identity between these two proteins, T cells recognize prominently the species-specific determinants localized within amino acid residues 21-40 and 49-72. Analysis of the molecular determinants of species-specificity for the M. leprae GroES sequence 25-40, using T-cell hybridomas and major histocompatibility complex (MHC)-binding assays, led to the identification of epitope cores and critical residues. Interestingly, closely overlapping epitope cores were found to be restricted by either H-2Ad (24-34) or H-2Ed (28-34). Furthermore, the site recognized by the M. leprae-specific monoclonal antibodies ML06 and ML10 was also localized in the overlapping sequences 25-31 and 25-29. In conclusion, we demonstrated that immunodominant species-specific T- and B-cell epitopes can be found in a mycobacterial heat-shock protein despite its highly conserved amino acid sequence. This finding suggests the feasibility of identifying a sufficient number of M. leprae-specific determinants for a composite T-cell immunodiagnostic reagent for tuberculoid leprosy.

[On the lesions caused by a leproma-derived and cultivated Mycobacterium HI-75 produced in ddY mice. With special reference to a factor influencing the lesions and the differences from those by BCG].

Not only in the experimental leprosy, primary aim to make every experimental model crucial for the medical research has been the simulation of the aspect of disease encountered in human case by the simplest possible way. The present study was conducted to do so making some variations in addition to the experimental lepromata, produced in nude mice by Sasaki et al. and by Hamit, utilizing a leproma-derived and cultivated Mycobacterium HI-75 (HI-75). In this study HI-75, Mycobacterium bovip BCG (BCG) and female SPF ddY(ddY) were utilized to make experimental models. In addition to these combinations, the effect of the immunization of beta-glucuronidase binding protein (BGBP) to the lesion was also examined. The BGBP extracted from pisum sativum and utilized in this study shows cross-immunoreactivity with those of HI-75 and M. leprae. As the results, the lesions caused by HI-75 and BCG were somewhat resembling though HI-75 caused a little more extensive lesions especially in lymphocytic and monocytic infiltration. Also HI-75 caused distinct nerve lesions(NL) in which the bacilli were often encountered in the endoneurium but not in those by BCG. Contrarily in mice immunized with BGBP, the lesions were only a little milder and the affected tissue was a little fibrosed. However, in NL the solid form HI-75 were more often observed in the endoneurium. The results indicated that the effect of BGBP immunization on the HI-75 induced lesion was not very clear by the present study alone, however, the proposed models itself should be and will become very useful, for experimental leprology with only slight modifications.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

Infection of SCID mice with Mycobacterium leprae and control with antigen-activated "immune" human peripheral blood mononuclear cells.

The SCID (severe combined immunodeficient) mouse lacks both B and T cells and tolerates injected mononuclear cells from humans, the principal hosts of Mycobacterium leprae. A SCID mouse model of leprosy could be useful to investigate potential vaccine strategies using human cells in a context in which the growth of the organism is monitored. Initial experiments determined that SCID mice are more susceptible than normal mice to infection and dissemination of M. leprae. Cells from humans, either BCG vaccinated or from countries where leprosy is endemic, were stimulated in vitro with a number of mycobacterial antigens--whole M. leprae, M. leprae cell walls, purified protein derivative of M. tuberculosis, and Mycobacterium bovis BCG--and tested for proliferation and production of interleukin-6, tumor necrosis factor alpha, and gamma interferon. Cell walls were the most efficient and consistent in inducing all of these activities. In vitro-activated human cells retain function better after injection into SCID mice than nonactivated cells. To test the ability of cells to affect the growth of M. leprae in the footpads of SCID mice, cells from a known responder to mycobacterial antigens and from a nonresponder were activated by M. leprae cell wall antigens. The cells were harvested and coinjected with fresh M. leprae into the right hind footpads of SCID mice. After 3 months, there was no growth of M. leprae in the footpads of mice coinjected with cells from the mycobacterial antigen responder, while growth was uninhibited in mice receiving cells from the nonresponder. Future experiments will determine requirements for antigen specificity in inhibiting M. leprae multiplication.

Analysis of the Mycobacterium tuberculosis 85A antigen promoter region.

A mycobacterial expression-secretion vector was constructed in which the Escherichia coli alkaline phosphatase (phoA) reporter gene was placed under the control of the Mycobacterium tuberculosis 85A promoter and secretion signal sequences. In recombinant Mycobacterium smegmatis and Mycobacterium bovis BCG, PhoA activity could readily be detected on the mycobacterial cell surface and in the culture supernatant, indicating that the 85A signals can drive heterologous expression and secretion in both species. In contrast to the mycobacteria, the 85A promoter did not function in E. coli. We mapped the promoter region by progressive deletions using BAL 31 exonuclease and by primer extension analysis. Insertion and deletion mutations within the promoter region indicated that, unlike most E. coli promoters but similar to Streptomyces promoters, the position of the putative -35 region was not critical for efficient promoter activity. In addition, we investigated the ability of the identified signals to drive the production and secretion in BCG of recombinant Schistosoma mansoni glutathione S-transferase (Sm28GST), a protective antigen against schistosomiasis. BALB/c mice immunized with the recombinant BCG by a single dose exhibited a weak but specific T-cell response to Sm28GST.

Identification of T cell stimulatory epitopes from the 18 kDa protein of Mycobacterium leprae.

We have used different mouse strains to examine in vivo and in vitro responses to the 18 kDa protein of Mycobacterium leprae, which appears to be strongly immunogenic in both mice and humans. B and T cell stimulatory epitopes recognised by different strains of mice have been mapped using overlapping peptides that span the entire 18 kDa protein. Previous work established that immunization of mice with the 18 kDa protein results in specific antibody production to common B cell epitopes and immunization of mice with peptides containing these B cell epitopes resulted in the induction of specific IgG to only a limited subset of epitopes in each strain. Now we report that T cells purified from mice immunized with peptides that stimulate antibody production, proliferate in vitro when rechallenged. The proliferating T cells produce levels of IL-2 and IFN-gamma, that indicate antigen-specific T helper type 1 cells are present in significant numbers. Thus, a comparison of in vivo and in vitro data suggests that T cells bearing the phenotype associated with potentially protective cell-mediated responses can be primed in vivo by epitopes on small peptides. Since T cells from both strains of mice are capable of responding to the immunogenic synthetic peptides in vitro, but give different responses to the same peptides in vivo, factors other than epitope structure appear to influence T cell subset activation. This may have important implications for diseases such as leprosy where a polarized T cell response appears to develop and for the development of synthetic subunit vaccines.

Major histocompatibility complex non-restricted presentation to CD4+ T lymphocytes of Mycobacterium leprae heat-shock protein 65 antigen by macrophages transfected with the mycobacterial gene.

When the immunodominant 65,000 MW heat-shock protein of Mycobacterium leprae (ML65hsp) was expressed from the transfected mycobacterial gene in the mouse macrophage cell line J774.G8, the antigen was recognized by specifically sensitized CD4+ splenocytes in association with major histocompatibility complex (MHC) class II and CD4. Inhibition by monensin, leupeptin and chloroquine but not brefeldin A indicated dependence of presentation upon endosomal antigen processing. Although direct access of the endogenously synthesized antigen to the endosomal pathway of presentation, without extracellular release followed by endocytosis, could not be discounted, antigen was present in supernatants of the transfected cells in a form that could be presented by fixed macrophages and a form that required further processing for presentation. Each of three monoclonal antibodies (mAb) specific for widely separated linear amino acid epitopes of the antigen strongly inhibited recognition, suggesting steric interference with antigen-presenting cell (APC)-T cell interaction. Tests with splenocytes from vaccinated congenic mice indicated that recognition was not restricted by MHC haplotype. The significance and mechanism of this apparent MHC context-independent interaction of the presented antigen with specific T-cell receptor (TcR) remain to be explored.

Evidence for B-lymphocyte mitogen activity in Borrelia burgdorferi-infected mice.

Borrelia burgdorferi produces a mitogen for murine B lymphocytes which can be measured in vitro by polyclonal stimulation of proliferation and immunoglobulin production (R. Schoenfeld, B. Araneo, Y. Ma, L. Yang, and J. J. Weis, Infect. Immun. 60:455-464, 1992). Sonicated B. burgdorferi cells also stimulated IL-6 production by splenocyte cultures. We have used the murine model for Lyme disease described by Barthold et al. (S. W. Barthold, D. S. Beck, G. M. Hansen, G. A. Terwilliger, and K. D. Moody, J. Infect. Dis. 162:133-138, 1990) to determine whether the B. burgdorferi B-cell mitogen is expressed during active infection. To correlate arthritic changes with immune events, we have studied two strains of mice injected with B. burgdorferi; one of them, C3H/HeJ, developed severe disease, and the other, BALB/c, developed only mild disease. C3H/HeJ mice displayed a persistent 10-fold increase in circulating immunoglobulin G (IgG) levels, a 2-fold increase in IgM levels, and a 15-fold increase in peripheral lymph node B-cell numbers, providing evidence of mitogenic activity. Infected BALB/c mice also had evidence for mitogen activity, since the IgG level in serum increased three- to fourfold. The bulk of the increase in circulating IgG levels was not directed against B. burgdorferi antigens, supporting the occurrence of polyclonal B-cell activation. Analysis of IgG isotypes pointed out a contrast between C3H/HeJ and BALB/c mice in that levels of all isotypes were elevated somewhat in both strains of infected mice but IgG2a levels were much more dramatically increased in the C3H/HeJ mice (28-fold) than in the BALB/c mice (4-fold). In this study, interleukin-6 levels were found to be persistently elevated in the serum of infected C3H/HeJ mice. Interestingly, interleukin-6 levels in serum were much lower in the infected BALB/c mice. These findings indicate that the B. burgdorferi mitogen is active in infected animals and may contribute to the inflammatory and immune response to infection.

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.