Mode of transmission and histology of M. leprae infection in nude mice

Athymic (nude) mice were experimentally infected with Mycobacterium leprae via the alimentary and respiratory tracts and through the skin. Animals were allowed to inhale aerosols of M. leprae or had bacilli instilled into the nostrils or directly into the lungs. Others were fed M. leprae by gastric tube or had bacilli placed on the tongue. Attempts were also made to transmit M. leprae from infected footpads by Aedes aegyptii mosquitoes. The most successful infections resulted from nasal instillations and from bacilli inoculated onto the tongue surface: in these cases heavy systemic infections occurred. M. leprae was also shown to survive passage through the alimentary tract and bacilli recovered from the faeces were capable of causing infection in recipient nude mice. The possible epidemiological significance of these findings for the transmission of leprosy in man is discussed.     

Superinfection in mice previously infected with Mycobacterium leprae.

Previous studies of the protection of mice by prior infection with Mycobacterium leprae in one hind footpad against challenge with M.leprae in the opposite hind footpad had produced conflicting results; therefore, the problem was restudied. In several experiments, BALB/c mice were inoculated first in the right hind footpad with 5,000 M. leprae and then challenged in the left hind footpad with 5,000 M. leprae of the same strain at intervals after primary infection, at the same time that uninfected mice were inoculated. Multiplication of the M. leprae of the secondary challenge inoculum occurred at the same rate and to the same level as multiplication in uninfected mice when challenges were made soon after primary infection. Multiplication was slowed but proceeded to the same level in previously infected as in uninfected mice when the challenges were administered between 76 and 106 days after primary infection (47 to 17 days before the M. leprae of the primary inoculum had multiplied to the level of 10-6 organisms per footpad). Finally, the M. leprae of a secondary challenge administered at the time that the organisms of the primary inoculum had multiplied to 10-6 per footpad or later not only multiplied more slowly in previously infected than in control animals, but multiplication in the previously infected animals reached a lower maximum. These results are similar to those observed when mice previously infected with M. bovis (BCG), M. marinum, Toxoplasma gondii, or Besnoitia jellisoni were challenged with M. leprae.     

Adoptively transferred dendritic cells restore primary cell-mediated inflammatory competence to acutely malnourished weanling mice

Immune depression associated with prepubescent malnutrition underlies a staggering burden of infection-related morbidity. This investigation centered on dendritic cells as potentially decisive in this phenomenon. C57BL/6J mice, initially 19 days old, had free access for 14 days to a complete diet or to a low-protein formulation that induced wasting deficits of protein and energy. Mice were sensitized by i.p. injection of sheep red blood cells on day 9, at which time one-half of the animals in each dietary group received a simultaneous injection of 10(6) syngeneic dendritic cells (JAWS II). All mice were challenged with the immunizing antigen in the right hind footpad on day 13, and the 24-hour delayed hypersensitivity response was assessed as percentage increase in footpad thickness. The low-protein diet reduced the inflammatory immune response, but JAWS cells, which exhibited immature phenotypic and functional characteristics, increased the response of both the malnourished group and the controls. By contrast, i.p. injection of 10(6) syngeneic T cells did not influence the inflammatory immune response of mice subjected to the low-protein protocol. Antigen-presenting cell numbers limited primary inflammatory cell-mediated competence in this model of wasting malnutrition, an outcome that challenges the prevailing multifactorial model of malnutrition-associated immune depression. Thus, a new dendritic cell-centered perspective emerges regarding the cellular mechanism underlying immune depression in acute pediatric protein and energy deficit.     

Adoptively transferred anti-idiotype pulsed B cells mediate autoimmune myocarditis.

Syngeneic mice receiving adoptively transferred enriched B cells or splenocytes pulsed in vitro with polyclonal anti-idiotypic antibodies (anti-Ids) against anti-CVB3 virus antibodies developed coxsackievirus B3 antigen-binding and virus-neutralizing antibodies during a 30-day period, in addition to overt expression of autoimmune myocarditis. Adoptive transfer of anti-Id pulsed T cells resulted in delayed appearance and transient expression of antiviral antibodies, and antiviral antibodies were marginal to absent in syngeneic animals receiving anti-Id pulsed macrophage populations. Proliferation analysis of recipient splenocytes indicated lack of proliferative capacity in response to monoclonal or polyclonal anti-Ids and only marginal proliferative capacity in response to coxsackievirus B3 virus antigen(s). In vitro assessment of delayed hypersensitivity in recipient animals demonstrated some specific immunity to anti-Ids in recipients receiving splenocytes or T cells. Anti-Ids expressing mimicry for heart-associated and/or viral antigen(s) interacting with B cells or other accessory cells suggest an autoantibody or anti-Id triggering of B-cell-mediated mechanisms involved in the development of myocarditis.     

Immunity to Mycobacterium leprae infections in mice stimulated by M. leprae, BCG, and graft-versus-host reactions.

Infections of mice with Mycobacterium leprae in one rear foot pad immunized them against a second infection in the other rear foot pad. Purified bacilli harvested from the first infection also produced immuniy when injection into the foot pads of previously uninfected mice. Injections of BCG afforded similar protection, but had no adjuvant effect on M. leprae. M. duvali, a cultivable mycobacterium that is reported to be more closely related antigenically to M. leprae than BCG is, provided much less protection against M. leprae challenge than BCG did. Moreover, when M. duvali was mixed with BCG, it was not any more effective than BCG alone. Graft-versus-host reactions, induced by injections of parental spleen cells into F1 hybrids, provided no protection against M. tuberculosis and M. marinum challenge. They gave moderate protection against M. leprae in one experiment but not in another with a different schedule. Allogenic spleen cells had a protective effect when injected locally into the infected foot pad. The effect produced by these injections of spleen cells was a delay in the appearance of bacterial growth; however, there was no decrease in the rate of logarithmic growth when it did appear and no reduction in the eventual plateau level.     

Monoclonal antibody immunotherapy in nude mice persistently infected with Cryptosporidium parvum.

Three groups of congenitally athymic nude mice were persistently infected following oral administration of 2 x 10(7) Cryptosporidium parvum oocysts. Two groups were treated once daily for 10 days with either neutralizing monoclonal antibody (MAb) 17.41 or an isotype control MAb. The third group received no treatment. Intestinal-infection scores were significantly decreased in nude mice treated with MAb 17.41 compared with isotype control MAb-treated and nontreated control groups (P less than 0.005). Biliary and pancreatic cryptosporidial-infection scores were similar for the MAb 17.41-treated and isotype control MAb-treated groups (P greater than 0.05).     

Mycobacterium leprae infection in nude mice: bacteriological and histological responses to primary infection and large inocula.

Previous studies have demonstrated that congenitally athymic, nude mice are highly susceptible to infection with Mycobacterium leprae. In this study, we showed that footpad inoculation of nude mice with different inoculum sizes of M. leprae resulted in exponential growth of bacilli until bacillary numbers reached approximately 10(10) bacilli per footpad. There was dissemination of the infection from approximately 10 months after inoculation. When nude mice were compared with thymectomized and irradiated mice and normal intact mice for the ability to detect growth from large inocula of low viability, nude mice were the most sensitive, permitting the detection of 10(2) viable M. leprae among 10(7) irradiation-killed organisms. There was widespread dissemination of the infection throughout the reticuloendothelial system and the tissues of the cooler body sites from approximately 10 months after inoculation. Histologically, the lesions resembled those seen in lepromatous leprosy, although the bacillary load appeared larger and was similar to that seen in heavily infected tissues of the nine-banded armadillo. An unusual feature was the presence of numerous foci of neutrophil polymorphs in the footpads and liver of infected nude mice.     

Vaccination with pure Mycobacterium leprae proteins inhibits M. leprae multiplication in mouse footpads.

In this study, we evaluated vaccination with a number of purified, as well as recombinant, Mycobacterium leprae proteins for protective efficacy in mice. BALB/c mice were immunized intradermally with various native somatic (purified) or recombinant M. leprae proteins and their synthetic polypeptides emulsified in Freund's incomplete adjuvant. The protective efficacy of these preparations was assessed by enumeration of bacilli in the footpads of mice challenged with viable M. leprae 1 to 2 months following immunization. Protection was afforded by the purified and recombinant 10-kDa M. leprae cytoplasmic heat shock protein, the recombinant cell wall-associated 65-kDa M. leprae heat shock protein, and to a lesser extent, the purified 28-kDa M. leprae cytoplasmic protein (superoxide dismutase). Vaccination with either the purified or recombinant 35-kDa M. leprae cell membrane protein, the synthetic 27-amino-acid N-terminal peptide of the 10-kDa protein, the recombinant 18-kDa M. leprae protein, or the purified 22-kDa cell membrane protein was ineffective. When the interval between immunization and challenge was increased to 6 months, the purified 10-kDa M. leprae protein and the recombinant 65-kDa M. leprae protein lost vaccine efficacy, while a sodium dodecyl sulfate-soluble protein fraction of the M. leprae cell wall (soluble proteins), as had been found previously, continued to protect, suggesting that multiple M. leprae protein epitopes are critical for solid vaccine protection.     

Induction of unresponsiveness to gamma interferon in macrophages infected with Mycobacterium leprae.

We have previously demonstrated that Mycobacterium leprae-burdened granuloma macrophages isolated from infected nude mice are refractory to activation by gamma interferon (IFN-gamma). To explore further both the afferent and efferent functional capacity of M. leprae-infected macrophages, we examined the IFN-gamma-mediated activation of resident mouse peritoneal macrophages infected in vitro with live or dead M. leprae. When IFN-gamma was administered within 24 h of M. leprae infection, macrophages were fully activated. However, defective activation was evident at 3 to 5 days postinfection in macrophages that were heavily burdened with viable M. leprae. This defect was evident by four parameters of activation in which IFN-gamma failed to stimulate the enhancement of microbicidal activity, cytotoxicity for tumor target cells, O2- production, and surface Ia antigen expression. The development of defective activation closely followed an increase in macrophage production of prostaglandin E2. Defective activation of M. leprae-burdened macrophages was reversible by indomethacin, and a similar block in IFN-gamma activation was observed in three of these four parameters in normal macrophages treated with exogenous prostaglandin E2. Thus, infection of mouse macrophages with M. leprae appears to restrict IFN-gamma-mediated activation at least in part by induction of inhibitory levels of prostaglandin E2.     

Airborne infection with Mycobacterium leprae in mice.

Although the portal of entry and mode of spread of M. leprae in human leprosy are still uncertain, it is widely held that direct person-to-person skin contact is important. This assumption has ignored the fact that patients with highly bacilliferous leprosy have nasal as well as dermal infection and that, since M. leprae is shed predominantly from the nose, leprosy might be an airborne infection. The present study was designed to investigate this possibility with mice exposed to airborne infection with M. leprae. The conditions are described in which thymectomised-irradiated CBA strain mice exposed to M. leprae aerosols sustained an immediate lung retention of 1 X 10(5) bacteria. Fourteen to 24 months later, 33% (10 of 30) of the mice had countable numbers of acid-fast bacilli (greater than 2 X 10(4)) with the characteristics of M. leprae in one or more homogenates prepared from ears, foot pads, nose or lungs. Evidence is presented from the distribution of M. leprae that the infection had arisen from systemic spresd of bacilli initially entering the lungs rather than from multiplication of organisms locally retained there, or in the nose, at the time of airborne infection. The relevance of these results to the possible route of infection of leprosy in man is discussed.     

Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum.

Dietary L-arginine (4%) significantly reduced fecal oocyst shedding in athymic nude mice chronically infected with Cryptosporidium parvum. This effect appeared to be due to an increase in host nitric oxide (NO) production as it was not observed in arginine-supplemented animals administered the NO synthase inhibitor, N-nitro-L-arginine methyl ester. N-Nitro-L-arginine methyl ester alone significantly increased fecal oocyst shedding in chronically infected animals. In in vitro assays, oocyst excystation and sporozoite viability were significantly reduced by the NO donors sodium nitroprusside and S-nitroso-L-acetyl penicillamine in a concentration-dependent manner. These data suggest that arginine-derived NO may reduce the parasite load in experimental cryptosporidiosis.     

Vaccination of mice with a soluble protein fraction of Mycobacterium leprae provides consistent and long-term protection against M. leprae infection.

Groups of BALB/c mice were vaccinated intradermally with either Freund's incomplete adjuvant (FIA) alone, 10(7) heat-killed Mycobacterium leprae organisms in FIA, or a number of fractions of M. leprae containing soluble and/or cell wall components. At 1, 3, 6, 9, and 12 months later, vaccinated mice were challenged in the right hind footpad with 5,000 live M. leprae organisms, and vaccine protection was assessed 6 to 8 months later, at the peak of M. leprae multiplication in the negative control (FIA alone), by the two-sample rank-sum test. In these studies, a cell wall fraction rich in peptidoglycan was consistently ineffective. Both heat-killed M. leprae and a fraction containing cell wall and fixed proteins generally protected when the interval between vaccination and challenge was 1 or 3 months but not subsequently. On the other hand, soluble proteins of M. leprae alone or in combination (with cell wall fractions) consistently (14 of 14 instances) afforded highly significant protection (P less than or equal to 0.01) at all challenge intervals up to 1 year after vaccination. These results suggest that the soluble protein fraction of M. leprae offers promise for a vaccine against leprosy.     

SDS-PAGE analysis of M. leprae protein antigens reacting with antibodies from sera from lepromatous patients and infected armadillos.

Studies have been conducted to characterize M. leprae bacilli derived from infected armadillos. First, the proteins of the mycobacterial extracts were fractionated by SDS-PAGE. Subsequently, the proteins in the gel were electrophoretically transferred on a strip of nitrocellulose paper by the technique of 'electrophoretic blotting'. The separated bacterial protein bands, thus immobilized on the nitrocellulose paper were made to react immunologically with sera from the lepromatous patients, infected armadillo sera and other experimental mycobacterial antisera. It was observed that a majority of M. leprae proteins contained antigenic determinants also present on proteins of BCG. In addition, only two specific antigen bands of 33KD and 12KD were conspicuously detected by the patients' sera and the infected armadillo sera. These substances were further identified as polysaccharides or glycoproteins since they could only be stained by Schiff's reagent or alcian blue. Only 12KD glycoprotein band reacted with concanavalin A, whereas wheat germ agglutinin (WGA) did not show any reaction with them. These 33KD and 12KD glycoprotein antigens were found to lose their antigenicity after pepsin treatment and can be considered as glycoproteins. Further, radiolabelling experiments showed that 12KD antigen underwent radioiodination under usual conditions, but 33KD glycoprotein failed to be similarly radiolabelled. It is suggested that these protein antigens have M. leprae specific determinants on a cross-reacting component.     

Cell-mediated resistance to infection with Listeria monocytogenes in nude mice.

Congenitally dysthymic nude (nu/nu) NMRI mice showed increased resistance to viable Listeria monocytogenes cells during the initial phase of infection as compared with euthymic control mice. The intravenous mean lethal dose (LD50), as determined for euthymic mice after an observation time of 7 and 14 days, amounted consistently to 6 X 10(4) Listeria. The corresponding values determined in nude mice were found to be increased by either 20-fold (1.2 X 10(6) Listeria after an observation time of 7 days) or 4-fold (2.4 X 10(5) Listeria after an observation time of 14 days). The transfer of spleen cells from immune euthymic donor mice into chronically infected nude mice caused almost complete elimination of Listeria within 1 week. The injection of dextran sulfate 24 h before a secondary infection with L. monocytogenes caused loss of antibacterial resistance in both chronically infected nude mice and Listeria-immune euthymic mice, this being expressed by a rapid increase in the numbers of bacteria in the spleens as well as the occurrence of serious signs of illness.     

Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reflects infection with or exposure to M. leprae

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-gamma) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-gamma secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-gamma secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.     

Studies on tumorigenicity of cells persistently infected with vesicular stomatitis virus in nude mice

Previous work has demonstrated that persistent infection of cultured cells (normally tumorigenic in the nude mouse) by any of a number of enveloped RNA viruses results in the loss of their ability to form tumors in nude mice. From the BHK 21/VSV persistently infected cell line which did not form tumors in these nude mice, we selected in vivo a subline which is tumorigenic even though it is still persistently infected. We report here a preliminary characterization of this subline. These virus-infected cells were consistently tumorigenic for more than six sequential passages in nude mice. They shed large amounts of virus and DI particles into the bloodstream and tissues of the mice; however, we found no evidence of the virus establishing a persistent infection in the mouse tissues. We demonstrated that virus isolated from this tumorigenic cell line was able to establish new persistently infected cell lines which were also tumorigenic, indicating that this is a characteristic of the virus. T₁ oligonucleotide mapping of the virion RNA recovered from this tumorigenic carrier subline demonstrates that the viral genome is undergoing continual mutation in vivo. This is similar to mutational changes occurring in vitro in the parental persistently infected cells. Very recently, the BHK 21 cell line persistently infected with VSV for over 5 years has spontaneously (with no in vivo selection) acquired the ability to form tumors in nude mice. In contrast to the other tumorigenic subline, this carrier line produces almost no mature virus or DI particles at present, and its tumorigenicity appears to be associated with very low virus expression in infected cells. Apparently, viruses can escape the natural killer (NK) cell response by a variety of means.     

Presence of Human T-Cell Responses to the Mycobacterium leprae 45-Kilodalton Antigen Reflects Infection with or Exposure to M. leprae

The ability of the 45-kDa serine-rich Mycobacterium leprae antigen to stimulate peripheral blood mononuclear cell (PBMC) proliferation and gamma interferon (IFN-γ) production was measured in leprosy patients, household contacts, and healthy controls from areas of endemicity in Mexico. Almost all the tuberculoid leprosy patients gave strong PBMC proliferation responses to the M. leprae 45-kDa antigen (92.8%; n = 14). Responses were lower in lepromatous leprosy patients (60.6%; n = 34), but some responses to the 45-kDa antigen were detected in patients unresponsive to M. leprae sonicate. The proportion of positive responses to the M. leprae 45-kDa antigen was much higher in leprosy contacts (88%; n = 17) than in controls from areas of endemicity (10%; n = 20). None of 15 patients with pulmonary tuberculosis gave a positive proliferation response to the 45-kDa antigen. The 45-kDa antigen induced IFN-γ secretion similar to that induced by the native Mycobacterium tuberculosis 30/31-kDa antigen in tuberculoid leprosy patients and higher responses than those induced by the other recombinant antigens (M. leprae 10- and 65-kDa antigens, thioredoxin, and thioredoxin reductase); in patients with pulmonary tuberculosis it induced lower IFN-γ secretion than the other recombinant antigens. These results suggest that the M. leprae 45-kDa antigen is a potent T-cell antigen which is M. leprae specific in these Mexican donors. This antigen may therefore have diagnostic potential as a new skin test reagent or as an antigen in a simple whole-blood cytokine test.     

Antibody response to phenolic glycolipid I in inbred mice immunized with Mycobacterium leprae.

The level of circulating antibody to phenolic glycolipid I of Mycobacterium leprae was determined in 18 inbred strains of mice after immunization with M. leprae organisms. By using a solid-phase radioimmunoassay with phenolic glycolipid I as test antigen, a continuous distribution of antibody levels ranging from high to low was observed. The level was found to be controlled by multiple genes, including both H-2 complex- and Igh allotype complex-linked genes. Low antibody response to phenolic glycolipid I was shown to be inherited as a dominant trait in three combinations of high X low responder F1 progeny.