Evolution of lymphocyte populations in armadillos (Dasypus novemcinctus) inoculated with M. leprae.

In human leprosy patients there are changes in the percentages of T and B lymphocytes in peripheral blood, and there is a correlation with the clinical characteristics or manifestations of the disease. These phenomena still require clarification regarding the triggering mechanism involved that may lead to one or the other clinical entities. Much has yet to be learned about the intricacies of whether the changes in subpopulations of T and B lymphocytes are a causative factor or an effect attributable to the microorganism itself. The armadillo is an excellent animal model to study how Mycobacterium leprae spread, turning into an established infection. The application of modifications in percentages of the subpopulations of B and T lymphocytes in armadillos may well lead to extrapolation of the results obtained in this animal model in an attempt to be able to manipulate the course of the disease in humans. The purpose of the study was to evaluate changes in the percentages of rosette-forming and sIgM+ mononuclear cells during a full year in groups of armadillos: five randomly chosen animals formed the control group and 11 armadillos were inoculated with M. leprae obtained from a human leproma at the onset of the 12-month period of the study. Of the 11 randomly selected armadillos that were inoculated, only five developed an active and disseminated infection. The percentage of rosette-forming cells did not show statistically significant variations during the first 6 months of the study. However, at months 8 and 12 a significant increment in this parameter was observed (p < 0.05) in the animals with active infection. In regard to the variations in the numbers of sIgM+ cells, significant changes occurred in the armadillos with active infection at month 2. However, results returned to normal and no changes were seen at later times. No significant changes occurred in the group of animals inoculated but not developing active infection compared with the other groups. The results are considered sufficiently interesting to encourage further study on the cell-mediated immune system of the armadillo and the changes that occur during the development and dissemination of an inoculated infection with M. leprae. Since this mammal is of great value as an effective animal model in the experimental research of M. leprae, there is an urgent need to obtain, as quickly as possible, a thorough understanding of the cellular branch of its immune system and, thereby, be in a position to extrapolate immune modulation to benefit human leprosy patients.

Immune response of armadillos (Dasypus novemcinctus). I. Use of lectins to identify lymphocyte subpopulations and to evaluate cell proliferation.

Lectins have been used to study populations and discrete differentiation stages of lymphocytes. Likewise, lectins have been of practical importance in promoting mitogenic stimulation of lymphocytes in numerous species. In this research project, we took advantage of these tools in an attempt to identify specific subsets of peripheral blood lymphocytes obtained from healthy nine-banded armadillos (Dasypus novemcinctus). The same cell source served to evaluate mitogenic stimulation. Twelve FITC-labeled lectins were used; 5 (ConA, LcH, RCA, WGA and UEA) reacted with almost 100% of the lymphocytes and 7 (PNA, DBA, SBA, PCA, PHA-L, PWM and VVA) recognized variable percentages (< 100% of these cells). This latter group of lectins may be useful in the identification of armadillo lymphocyte subsets, or may correlate with discrete stages of differentiation of these cells. The same lectins served to evaluate mitogenic stimulation in an aliquot of the same peripheral blood mononuclear cells. Of the 12 lectins studied, 5 (ConA, PHA-L, PWM, DBA and SBA) had the capacity to induce mitogenic stimulation in the whole mixture of mononuclear cells, giving rise to variable degrees in the corresponding mitogenic index obtained for each of the 5 lectins. Those lectins that gave an indication of selective identification of lymphocytes, that is, the percentages at or below 75%, may prove useful in the evaluation of the immune response of healthy armadillos as well as the evolution of progression stages of lepromatous leprosy in armadillos inoculated with the same strain of Mycobacterium leprae that affects humans.

Identification and purification of armadillo (Dasypus novemcinctus) immunoglobulins: preparation of specific antisera to evaluate the immune response in these animals.

In this work we describe the purification and characterization of armadillo immunoglobulins. The IgM was precipitated using low-strength ionic solution and further purified by filtration through Sephadex G-200. The IgG was obtained in pure form by precipitation of serum with ammonium sulfate and DEAE-cellulose ion exchange chromatography. The purity of these immunoglobulins was evaluated by polyacrylamide gel electrophoresis. The results showed 28-kDa light chains and 55-kDa and 70-kDa heavy chains for IgG and IgM, respectively. The rabbit antibodies against these molecules were used to prepare fluorescein (FITC) and peroxidase conjugates. The FITC conjugate was used to quantify IgM-bearing lymphocytes. An average of 17% of peripheral blood lymphocytes were sIgM+ from 14 healthy animals. Additionally, in the same animals we quantified lymphocytes with the capacity to form rosettes with sheep red-blood cells; the average for this marker was 10%. Also, the production of crossreacting antibodies to BCG was evaluated in healthy and Mycobacterium leprae-inoculated animals using the peroxidase conjugates. All animals with active infection recognized BCG antigens.

Use of synthetic peptides corresponding to sequences of Mycobacterium leprae proteins to study delayed-type hypersensitivity response in sensitized guinea pigs.

In this work we report the synthesis of 10 peptides (P1-P10) corresponding to one or several segments of the amino acid sequence of proteins from Mycobacterium leprae: 65 kDa, 28 kDa, 18 kDa, and 28 kDa superoxide dismutase, recently renamed antigens 2L, 9L, 12L, and 14L, respectively. They were assayed in the guinea pig model for the induction of a delayed-type hypersensitivity response in M. leprae and BCG-sensitized animals. To sensitize the animals two schemes were used: either a single dose of 5 x 10(9) irradiated or autoclaved whole bacilli, or four weekly intramuscular injections each containing 500 micrograms of soluble extract of M. leprae (MLSE) in incomplete Freund's adjuvant. Because the second scheme used far too much antigen, we decided to use the first scheme for the experiments we report here. DTH reactions of sensitized animals were induced after 30 days with intradermal injections of 5 micrograms of MLSE and with each of the 10 peptides at three different concentrations: 250 micrograms, 100 micrograms, and 0.05 micrograms. All M. leprae-sensitized guinea pigs gave indurations of 10 mm or more with MLSE, which indicates that the animals were sensitized. None of them gave DTH indurations with 250 micrograms or 100 micrograms, but some of them had positive DTH reactions with the 0.05 micrograms doses of the synthetic peptides. This is most likely due to the fact that we have used an outbred strain of guinea pigs. The peptides were also tested at 0.05 micrograms in animals sensitized with BCG. P7 and P10 seem to be nonspecific peptides; the remaining peptides only induced DTH in the M. leprae-sensitized guinea pigs. P3 (segments 65-85 of the 65-kDa protein) induced a positive DTH in 58% of the tested animals. In other experiments, guinea pigs were sensitized with a single injection (500 micrograms) of each of the synthetic peptides. All animals, except those sensitized with P4 and P8, had positive DTH responses when the homologous peptide was used. Those sensitized with P2, P4, P5, P7, and P8 were able to produce indurations when MLSE was used for the induction of the DTH reaction.

Biochemical alterations in the serum of armadillos (Dasypus novemcinctus) infected with Mycobacterium leprae. A preliminary report.

Armadillos (Dasypus novemcinctus) were inoculated with Mycobacterium leprae isolated from lepromas taken from untreated lepromatous patients or from the spleen of an armadillo previously infected with human M. leprae. The effect of the infection on the serum levels of lactic dehydrogenase (LDH), alkaline phosphatase (AlkP), glutamate-oxalacetate (GOT) and glutamate-pyruvate (GPT) transaminases was investigated. In general, there was a good correlation between positive evidences of infection and alterations in the levels of LDH, GOT, and GPT. Although elevations in LDH levels were more striking, elevations in GOT and GPT levels were more consistent with the disease. When an absolute increase in the total LDH activity was not observed in a M. leprae-infected animal, an increase in the level of LDH isozyme V was still clearly evident. Serum levels of alkaline phosphatase were not affected by the disease. The ratio GOT/GPT (greater than 1.0) in the infected animals reflected and supported the chronic nature of the disease and the liver involvement. The enzymatic alterations are not, however, specific for leprosy.