[History for animal model of Hansen's disease and characteristics of leprosy in hypertensive nude rat].

The long search of an animal model for leprosy were carried out as many researchers since the Mycobacterium leprae discovery by Dr. Hansen in 1874. The remarkable results were left after the development of the foot-pad method by Dr. Shepard in 1960. The introduction of the T-R mouse and athymic (nude) mouse for leprosy research, alsospontaneity examples of Hansen's disease was reported to armadillo, chimpanzee and mangabay monkey, and it was confirmed that Hansen's disease was the zoonosis. Although, We have established a congenic hypertensive nude rat, SHR/NCrj-rnu (SHR.F344-Foxn(rnu)), carrying nude (rnu) and hypertension genes. SHR/NCrj-rnu rats obtained showed high susceptibility to M. leprae and showed a characteristic disease with a progressive pattern of leproma formation. Also this hypertensive nude rat strain produce high level of IL-10. Therefore, congenic hypertensive nude rat may be useful for an animal model to leprosy.

Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components.

BACKGROUND: The transmission of Mycobacterium leprae, the causative pathogen of leprosy, has been postulated to occur mainly through upper respiratory route rather than skin-to-skin contact via minor injuries. The M. leprae genome contains mce1A gene, which encodes a putative mammalian cell entry protein. However, to date, there have been no functional analyses of the M. leprae mce1A gene product. OBJECTIVE: The aim of this study was to elucidate a possible relationship between this transmission mechanism and the mce1A gene product. METHODS: We analyzed the cell uptake activity in vitro of polystyrene latex beads coated with a purified recombinant (r-) protein expressed by a 849-bp locus within the mce1A gene. RESULTS: The r-protein promoted uptake of the beads into human nasal epithelial cells derived from nasal polyps, human bronchial epithelial cell line, normal human dermal fibroblasts, normal human microvascular endothelial cells and normal human keratinocytes cultured at 0.01 mM extracellular calcium concentration [Ca]; no uptake occurred with keratinocytes cultured at 1.2mM [Ca]. CONCLUSION: These results suggest that the mce1A gene product can mediate M. leprae entry into respiratory epithelial cells as their natural target cells, which may be the main mode of transmission. Endothelial cells, on the other hand, may serve as the reservoir of the bacilli for long-term infection. The M. leprae Mce1A protein has potential important implications for mode of transmission and pathogenesis of leprosy.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG motifs) against Mycobacterium leprae multiplication in mice.

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.

Effect of hsp65 DNA vaccination carrying immunostimulatory DNA sequences (CpG Motifs) against mycobacterium leprae multiplication in mice

A DNA expressing hsp65 of Mycobacterium leprae (pACB/hsp65) was constructed by using a vector containing immunostimulatory DNA sequences (pACB). At 12 weeks post-immunization, spleen cells from BALB/cA mice immunized with pACB/hsp65, produced a significantly higher amount of IFN-gamma than mice immunized with pACB in the absence of any in vitro stimulation, and further enhanced its production upon secondary in vitro stimulation with M. leprae lysate and hsp65. On the other hand, while production of IL-12 was observed in mice immunized with pACB/hsp65 12 weeks before, the cytokine production was inhibited by in vitro secondary stimulation with M. leprae or hsp65. At 18 weeks post-immunization, the production of both IFN-gamma and IL-12 was apparently down-regulated, but that of IL-10 was up-regulated. IL-10 seemed to suppress the IFN-gamma and IL-12 productions, because their production was recovered by neutralization of IL-10 with anti-IL-10 mAb. Furthermore, when the efficiency of pACB/hsp65 as a vaccine against M. leprae was evaluated in vivo, the mice immunized with pACB/hsp65 suppressed the multiplication of subsequently challenged M. leprae. These results suggest that a DNA containing M. leprae-derived hsp65 and immunostimulatory sequences might be a potent vaccine candidate against M. leprae infection.

Prevention of diabetes in non-obese diabetic mice by a single immunization with Mycobacterium leprae.

The incidence of overt diabetes was completely prevented by a single intradermal inoculation of Mycobacterium leprae (M. leprae) into Non-obese diabetic (NOD) mice as young as 6-7 weeks. Partial prevention was also observed in cases when 65 kD heat-shock protein (hsp65) with Freund's incomplete adjuvant (FIA) was injected, and no prevention was observed by 38 kD with FIA immunization. Histological examination of pancreata demonstrated that control and M. leprae-immunized mice at 24 weeks of age developed the insulitis even though the number of lymphocytes infiltrated in the treated ones were less than the controls. However, later, at 47 weeks of age, even the immunized mice become to develop very severe insulitis. Thus, M. leprae-immunization did not prevent the incidences of insulitis. The spontaneous development of serum antibody against hsp65 and 38 kD protein preceded the onset of diabetes in NOD mice. Lymphocytes response, IFN-gamma and IL-10 production of splenocytes cultures stimulated with hsp65 were examined to clue the reasons for the prevention of IDDM incidence by M. leprae immunization. The spontaneous development of anti-hsp65 T lymphocytes preceded the outbreak of overt IDDM in control NOD mice, but also appeared in M. leprae immunized cases in which the IDDM incidence was prevented, and both control and M. leprae immunized groups produced IFN-gamma and IL-10 by stimulation with hsp65.

[Susceptibility to Mycobacterium leprae of congenic hypertensive nude rat (SHR/NCrj-rnu) and production of cytokine from the resident peritoneal macrophages].

We have established a congenic hypertensive nude rat strain, SHR/NCrj-rnu, carrying nude (rnu) and hypertension genes which was produced using females of the SHR/NCrj rat and males of the F344/NJcl nude rat by cross-intercross system for 12 generations. We demonstrated the susceptibility to M. leprae infection of SHR/NCrj-rnu rats as compared with F344/NJcl-rnu rats. SHR/NCrj-rnu rats were highly susceptible to M. leprae, and the SHR/NCrj-rnu rats of both sexes showed massive swelling of legs due to multiplication of M. leprae. However, F344/NJcl-rnu rats of both sexes revealed very poor susceptibility to M. leprae. There was a wide difference in the susceptibility to M. leprae between the SHR/NCrj-rnu and the F344/NJcl-rnu rats. We also examined the cytokine production. The resident peritoneal macrophages of SHR/NCrj-rnu rats produced IL-1 alpha, IL-6, IL-10 and TNF alpha, whereas those of F344/NJcl-rnu rats produced only TNF alpha.

Leprosy in hypertensive nude rats (SHR/NCrj-rnu)

Since more than a decade ago, we have attempted to develop spontaneously hypertensive rats carrying the nude gene that permits high multiplication of Mycobacterium leprae. A congenic strain carrying nude (rnu) and hypertensive genes was produced using SHR/NCrj females and F344/NJcl-rnu males. Cross-intercross was carried out 12 times to establish the hypertensive nude rat congenic strain. As a result of the genetic monitoring test with NE12F2 generation rats, the genetic profile of the SHR/NCrj-rnu rats was the same as that of the SHR/NCrj rats except for the rnu gene. We have successfully developed a hypertensive congenic nude rat strain (SHR.F344Hfh11; SHR/NCrj-rnu). An increase in the blood pressure in nude rats was found to begin at a slightly delayed age when compared with their hairy litter mates. Both female and male rats showed the highest blood pressure at approximately 20 weeks of age--166 +/- 1.4 and 197 +/- 11 mm Hg in nude rats and 175 +/- 11 and 193 +/- 3.2 mm Hg in their hairy litter mates in female and male rats, respectively. In the present study, comparisons were made on the susceptibility to M. leprae in hypertensive SHR/NCrj-rnu and normotensive F344/NJcl-rnu rats. We have reconfirmed that hypertensive SHR/NCrj-rnu rats of the NE12F3 generation were highly susceptible to M. leprae. In the SHR/NCrj-rnu rats of both sexes excellent massive swelling due to multiplication of M. leprae was observed and, also, nodular lesions were produced in uninoculated fore feet and lips while those sites in the F344/NJcl-rnu rats showed only a slight swelling of the inoculated feet with mild nodular lesions. Although mild lymphocyte proliferation was seen only in the M. leprae-inoculated site with numerous bacilli and partial necrosis in the SHR/NCrj-rnu rats, at noninoculated sites, multiplication of M. leprae was only observed in the cells of the mononuclear phagocyte system. However, in F344/NJcl-rnu rats, lymphocyte proliferation with a few neutrophils was seen at the site of inoculated hind foot pads and everywhere at the site of multiplication of M. leprae. There was a wide difference in the susceptibility to M. leprae between the SHR/NCrj-rnu and the F344/NJcl-rnu rats.

Susceptibility to Mycobacterium leprae of ALY (Alymphoplasia) mice and IFN-gamma induction in the culture supernatant of spleen cells

The aly/aly (alymphoplasia) mice from a mutation of a colony of the C57BL/6J mouse strain, which has a systemic absence of lymph nodes and Peyer's patches, are deficient in both T- and B-cell-mediated immune functions. We have undertaken a comparison of susceptibility to Mycobacterium leprae of ALY (aly/aly, aly/+) mice with C57BL/6J mice. The aly/aly mouse was found to have an excellent high susceptibility to M. leprae with no distinction between female and male. The aly/+ mouse also was more susceptible to M. leprae at an earlier stage than the C57BL/6J mouse. Therefore, we examined and compared the cytokine gene expression and gamma interferon (IFN-gamma) induction in the splenocytes of ALY mice. The expression of interleukin 4 (IL-4), IL-10 and IL-12 mRNA was weakly stimulated with ML-lysate in inoculated aly/aly mice but IL-2, IL-6, IGIF/IL-18 and IFN-gamma mRNA were not observed. None of the cytokine genes used appeared, except the mRNA for IL-1-alpha, when uninfected cultured spleen cells were stimulated with ML-lysate. Also, IFN-gamma production was not induced. However, the appearance of these cytokine genes was observed when stimulated with concanavalin A (ConA), and IFN-gamma production was also induced in the culture supernatant by aly/+ and even aly/aly mice stimulated with ConA. To examine the reason why IFN-gamma cannot be produced by splenocytes of ALY mice inoculated with M. leprae, we detected cytokine gene expression and IFN-gamma induction in the presence of recombinant murine IL-12 or IGIF/IL-18. IL-2 mRNA expression was detected in all of the mice tested in the presence of IL-12 but not in aly/aly mice under IGIF/IL-18, and iNOS mRNA expression was not observed in aly/aly mice under IL-12 or IGIF/IL-18. IL-4 and IL-10 mRNA were detected by aly/aly mice only by exposure to IGIF/IL-18. In culture, the supernatant with ML antigens of the aly/aly mice did not produce IFN-gamma in spite of the presence of IL-12 and IGIF/IL-18, while IFN-gamma was weakly induced in aly/+ mice stimulated with ML-lysate and in the presence of IGIF/IL-18. Nevertheless, IFN-gamma production was observed in splenocytes of the aly/aly mice stimulated with ConA and also with IGIF/IL-18 plus anti-CD3 antibody. Our results suggest that ALY mice might be showing a high susceptibility to M. leprae because of deficient priming for activation of T cells with the leprosy bacilli infection. Moreover, it is possible that the phagocytic activities of the macrophages of ALY mice are also impaired.

Cytokine gene expression in the foot pad and spleen of BALB/cAJcI mice infected with M. leprae

The cytokine mRNAs expressed in the foot pads and spleens of BALB/cAJcl mice infected with Mycobacterium leprae were studied by the reverse transcriptase-polymerase chain reaction (RT-PCR) method using cytokine-specific primers for interleukin-1 alpha (IL-1 alpha), -2, -4, -6, -10, -12-(p40), gamma interferon (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and TNF-beta, and then for CD4 and CD8 markers. The pattern of cytokine gene expression in the foot pad which supports M. leprae growth was different from the expression in the spleen which does not permit M. leprae multiplication in mice. Before BALB/cAjcl mice were infected with M. leprae, IL-1 alpha and TNF-beta mRNAs were expressed physiologically in the foot pad while all of the cytokine genes examined were expressed in the spleen. In the foot pads of mice inoculated with M. leprae, in addition to the physiological appearance of IL-1 alpha and TNF-beta mRNAs, these signals were intensified. TNF-alpha expression was induced by the infection. On the other hand, in the spleens of mice inoculated with M. leprae, CD4 mRNA expression disappeared on day 1 of the infection, which was accompanied by the reduced expression of IL-2, -4, -6, and -12 mRNAs. The recovery of CD4 mRNA expression at a latter stage was accompanied by a corresponding increase of the cytokine mRNA expression. It was suspected that these results might permit restricted growth of M. leprae in the foot pads of normal mice. Furthermore, our study suggests that tissue-specific, local, immunologic characteristics are important in M. leprae growth.